Long-acting veterinary polypeptides and methods of producing and administering same

ABSTRACT

A polypeptide and polynucleotides comprising at least two carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to a non-human peptide-of-interest are disclosed. Pharmaceutical compositions comprising the non-human polypeptides and polynucleotides of the invention and methods of using both human and non-human polypeptides and polynucleotides are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/702,156 filed on Feb. 5, 2007. U.S. application Ser. No. 11/702,156claims priority of U.S. Provisional Application Ser. No. 60/764,761,filed Feb. 3, 2006, which is hereby incorporated in its entirety byreference herein.

FIELD OF INVENTION

A polypeptide and polynucleotides encoding same comprising at least twocarboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to apeptide-of-interest are disclosed. Pharmaceutical compositionscomprising the polypeptide and polynucleotides of the invention andmethods of using same are also disclosed.

BACKGROUND OF THE INVENTION

Polypeptides are susceptible to denaturation or enzymatic degradation inthe blood, liver or kidney. Accordingly, polypeptides typically haveshort circulatory half-lives of several hours. Because of their lowstability, peptide drugs are usually delivered in a sustained frequencyso as to maintain an effective plasma concentration of the activepeptide. Moreover, since peptide drugs are usually administrated byinfusion, frequent injection of peptide drugs cause considerablediscomfort to a subject. Thus, there is a need for technologies thatwill prolong the half-lives of therapeutic polypeptides whilemaintaining a high pharmacological efficacy thereof. Such desirouspeptide drugs should also meet the requirements of enhanced serumstability, high activity and a low probability of inducing an undesiredimmune response when injected into a subject.

Unfavorable pharmacokinetics, such as a short serum half-life, canprevent the pharmaceutical development of many otherwise promising drugcandidates. Serum half-life is an empirical characteristic of amolecule, and must be determined experimentally for each new potentialdrug. For example, with lower molecular weight polypeptide drugs,physiological clearance mechanisms such as renal filtration can make themaintenance of therapeutic levels of a drug unfeasible because of costor frequency of the required dosing regimen. Conversely, a long serumhalf-life is undesirable where a drug or its metabolites have toxic sideeffects.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a polypeptidecomprising at least two chorionic gonadotrophin carboxy terminal peptide(CTP) attached to a non-human peptide of interest.

In another embodiment, the present invention provides a polypeptidecomprising a first chorionic gonadotrophin CTP attached to an aminoterminus of a non-human peptide of interest and a second chorionicgonadotrophin CTP attached to a carboxy terminus of a polypeptidesequence of interest.

In another embodiment, the present invention provides a polypeptide,comprising two chorionic gonadotrophin CTP attached to a carboxyterminus of a non-human peptide of interest.

In another embodiment, the present invention provides a polypeptidecomprising a first chorionic gonadotrophin CTP attached to an aminoterminus of non-human peptide of interest, and a second and thirdchorionic gonadotrophin CTP attached to a carboxy terminus of apolypeptide sequence of interest.

In another embodiment, the present invention provides a polypeptidecomprising at least three chorionic gonadotrophin CTP attached to anon-human peptide of interest.

In another embodiment, the present invention provides a polynucleotidecomprising a sequence encoding a polypeptide, comprising at least twochorionic gonadotrophin CTP attached to a non-human peptide of interest.

In another embodiment, the present invention provides a polynucleotidecomprising a sequence encoding a first chorionic gonadotrophin CTPattached to an amino terminus of non-human peptide of interest and asecond chorionic gonadotrophin CTP attached to a carboxy terminus of apolypeptide sequence of interest.

In another embodiment, the present invention provides a polynucleotidecomprising a sequence encoding two chorionic gonadotrophin CTP attachedto a carboxy terminus of non-human peptide of interest.

In another embodiment, the present invention provides a polynucleotidecomprising a sequence encoding a first chorionic gonadotrophin CTPattached to an amino terminus of non-human peptide of interest, and asecond and third chorionic gonadotrophin CTP attached to a carboxyterminus of a polypeptide sequence of interest.

In another embodiment, the present invention provides a polynucleotidecomprising a sequence encoding at least three chorionic gonadotrophinCTP attached to a non-human peptide of interest.

In another embodiment, the present invention provides a method oftreating or reducing the incidence associated with a growth,weight-related, or metabolic condition in a non-human subject,comprising administering to a subject a therapeutically effective amountof CTP-GH, thereby treating a non-human subject having a growth,weight-related, or metabolic condition. In another embodiment, the GHpeptide is a non-human GH peptide. In another embodiment, the GH peptideis a human peptide. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method ofimproving a biological half life of a non-human peptide of interest,comprising the step of attaching at least two chorionic gonadotrophinCTP sequences to the non-human peptide of interest, thereby improving abiological half life of a non-human peptide of interest.

In another embodiment, the present invention provides a method ofadministering a peptide of interest to a non-human subject in needthereof, comprising the step of attaching at least two chorionicgonadotrophin CTP sequences to the peptide of interest, therebyadministering a peptide of interest to a non-human subject in needthereof. In another embodiment, the peptide of interest is a non-humanpeptide of interest. In another embodiment, the peptide of interest is ahuman peptide of interest. Each possibility represents a separateembodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are diagrams illustrating six EPO-CTP constructs.

FIG. 1A—is a diagram of the polypeptide of SEQ ID NO: 1

FIG. 1B is a diagram of the polypeptide of SEQ ID NO: 2

FIG. 1C is a diagram of the polypeptide of SEQ ID NO: 3

FIG. 1D is a diagram of the polypeptide of SEQ ID NO: 4.

FIG. 1E is a diagram of the polypeptide of SEQ ID NO: 5.

FIG. 1F is a diagram of the polypeptide of SEQ ID NO: 6.

FIG. 2 is a photograph illustrating the expression of the EPO-CTPvariants from transfected DG44 cells. Final test samples fromtransfected cells were prepared as described under “sample preparation”and run on SDS/PAGE. Proteins were detected by western blot.

FIG. 3 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-3 (SEQ ID NO: 3). ICR mice (n=7/group) receiveda single IV injection/week (15 μg/kg) for three weeks of EPO-3, rhEPO-WT(SEQ ID NO: 16), Recormon (Commercial EPO) or Recormon (5 μg/kg) 3 timesa week. Control animals were injected IV with PBS. Blood samples werecollected three times a week and haematocrit levels were detected. Eachpoint represents the group average of haematocrit (%)±SE.

FIG. 4 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-1 (SEQ ID NO: 1). ICR mice (n=7/group) receiveda single IV injection/week (15 μg/kg) for three weeks of EPO-1, rhEPO-WT(SEQ ID NO: 16), Recormon or Recormon (5 μg/kg) 3 times a week. Controlanimals were injected IV with PBS. Blood samples were collected threetimes a week and haematocrit levels were detected. Each point representsthe group average of haematocrit (%)±SE.

FIG. 5 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-2 (SEQ ID NO: 2). ICR mice (n=7/group) receiveda single IV injection/week (15 μg/kg) for three weeks of EPO-2 (SEQ IDNO: 2), rhEPO-WT (SEQ ID NO: 16), Recormon or Recormon (5 μg/kg) 3 timesa week. Control animals were injected IV with PBS. Blood samples werecollected three times a week and haematocrit levels were detected. Eachpoint represents the group average of haematocrit (%)±SE.

FIG. 6 is a time graph illustrating the change in reticulocyte levelfollowing a single bolus dose of EP0-0 (SEQ ID NO: 16), EPO-3 (SEQ IDNO: 3) and Aranesp.

FIG. 7 is a time graph illustrating the change in hemoglobin level(presented as change from baseline) following a single bolus dose ofEP0-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp.

FIG. 8 is a time graph illustrating the change in hematocrit levelfollowing a single bolus dose of EP0-0 (SEQ ID NO: 16), EPO-3 (SEQ IDNO: 3) and Aranesp.

FIG. 9 is a graph illustrating the change in serum concentration ofEPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp post i.v.injection.

FIG. 10 is a Western blot illustrating the molecular weight & identityof MOD-4020 (SEQ ID NO: 36), MOD-4021 (SEQ ID NO: 37), MOD-4022 (SEQ IDNO: 38), MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40). PAGE SDSgel was blotted and stained using monoclonal anti-hGH antibodies. Thephotograph indicates that like commercial and wild type hGH, MOD-7020-4variants are recognized by anti-hGH antibodies.

FIG. 11 is a bar graph illustrating the weight gain of hypophysectomizedrats following administration of the GH-CTP polypeptides of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention describes long-actingpolypeptides and methods of producing and using same. In anotherembodiment, long-acting polypeptides comprise carboxy terminal peptide(CTP) of human Chorionic Gonadotropin (hCG). In another embodiment, CTPacts as a protectant against degradation of proteins or peptides derivedtherefrom. In another embodiment, CTP extends circulatory half-lives ofproteins or peptides derived therefrom. In some embodiments, CTPenhances the potency of proteins or peptides derived therefrom.

In another embodiment, “CTP peptide,” “carboxy terminal peptide,” and“CTP sequence” are used interchangeably herein. In another embodiment,the carboxy terminal peptide is a full-length CTP. In anotherembodiment, the carboxy terminal peptide is a truncated CTP. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, “signal sequence” and “signal peptide” are usedinterchangeably herein. In another embodiment, “sequence” when inreference to a polynucleotide can refer to a coding portion. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, “peptide of interest” and “polypeptidesequence-of-interest” are used interchangeably herein. In anotherembodiment, the peptide of interest is a full-length protein. In anotherembodiment, the peptide of interest is a protein fragment. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a polypeptide comprising at least twocarboxy-terminal peptide (CTP) sequences of chorionic gonadotrophinattached to a polypeptide sequence-of-interest, wherein a first CTPsequence of the at least two CTP sequences is attached to an aminoterminus of the polypeptide sequence of interest and a second CTPsequence of the at least two CTP sequences is attached to the carboxyterminus of the polypeptide sequence of interest is provided. In anotherembodiment, the carboxy-terminal peptide (CTP) sequence is of humanchorionic gonadotrophin.

In another embodiment, the carboxy-terminal peptide (CTP) is attached tothe polypeptide sequence of interest via a linker. In anotherembodiment, the linker which connects the CTP sequence to thepolypeptide sequence of interest is a covalent bond. In anotherembodiment, the linker which connects the CTP sequence to thepolypeptide sequence of interest is a peptide bond. In anotherembodiment, the linker which connects the CTP sequence to thepolypeptide sequence of interest is a substituted peptide bond.

The phrase “polypeptide sequence of interest” refers, in anotherembodiment, to any polypeptide sequence, such as one comprising abiological activity. In another embodiment, the peptide is glycosylated.In another embodiment, the peptide is non-glycosylated. Examples ofpolypeptides which benefit from an extension in their circulatoryhalf-life include, but are not limited to erythropoietin (EPO),interferons, human growth hormone (hGH) and glucagon-like peptide-1(GLP-1).

In another embodiment, the carboxy terminal peptide (CTP) of humanChorionic Gonadotropin (hCG) is fused to a protein. In anotherembodiment, the carboxy terminal peptide (CTP) of human hCG is fused toa glycoprotein. In another embodiment, the carboxy terminal peptide(CTP) of hCG is fused to a glycoprotein hormone. In another embodiment,the CTP of hCG is fused to a peptide derived from a glycoproteinhormone. In some embodiments, glycoprotein hormones comprise EPO, FSH,or TSH and peptides derived therefrom.

In some embodiments, a CTP sequences at both the amino terminal end of apolypeptide and at the carboxy terminal end of the polypeptide provideenhanced protection against degradation of a protein. In someembodiments, CTP sequences at both the amino terminal end of apolypeptide and at the carboxy terminal end of the polypeptide provideextended half-life of the attached protein.

In some embodiments, a CTP sequence at the amino terminal end of apolypeptide, a CTP sequence at the carboxy terminal end of thepolypeptide, and at least one additional CTP sequence attached in tandemto the CTP sequence at the carboxy terminus provide enhanced protectionagainst degradation of a protein. In some embodiments, a CTP sequence atthe amino terminal end of a polypeptide, a CTP sequence at the carboxyterminal end of the polypeptide, and at least one additional CTPsequence attached in tandem to the CTP sequence at the carboxy terminusprovide extended half-life of the attached protein. In some embodiments,a CTP sequence at the amino terminal end of a polypeptide, a CTPsequence at the carboxy terminal end of the polypeptide, and at leastone additional CTP sequence attached in tandem to the CTP sequence atthe carboxy terminus provide enhanced activity of the attached protein.

In some embodiments, a CTP sequence at the amino terminal end of apolypeptide, a CTP sequence at the carboxy terminal end of thepolypeptide, and at least one additional CTP sequence attached in tandemto the CTP sequence at the amino terminus provide enhanced protectionagainst degradation of the attached protein. In some embodiments, a CTPsequence at the amino terminal end of a polypeptide, a CTP sequence atthe carboxy terminal end of the polypeptide, and at least one additionalCTP sequence attached in tandem to the CTP sequence at the aminoterminus provide extended half-life of the attached protein. In someembodiments, a CTP sequence at the amino terminal end of a polypeptide,a CTP sequence at the carboxy terminal end of the polypeptide, and atleast one additional CTP sequence attached in tandem to the CTP sequenceat the amino terminus provide enhanced activity the attached protein.

In another embodiment, the carboxy terminal peptide (CTP) peptide of thepresent invention comprises the amino acid sequence from amino acid 112to position 145 of human chorionic gonadotrophin, as set forth in SEQ IDNO: 17. In another embodiment, the CTP sequence of the present inventioncomprises the amino acid sequence from amino acid 118 to position 145 ofhuman chorionic gonadotropin, as set forth in SEQ ID NO: 18. In anotherembodiment, the CTP sequence also commences from any position betweenpositions 112-118 and terminates at position 145 of human chorionicgonadotrophin. In some embodiments, the CTP sequence peptide is 28, 29,30, 31, 32, 33 or 34 amino acids long and commences at position 112,113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.

In another embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 1-5 conservativeamino acid substitutions as described in U.S. Pat. No. 5,712,122. Inanother embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 1 conservativeamino acid substitution. In another embodiment, the CTP peptide is avariant of chorionic gonadotrophin CTP which differs from the native CTPby 2 conservative amino acid substitutions. In another embodiment, theCTP peptide is a variant of chorionic gonadotrophin CTP which differsfrom the native CTP by 3 conservative amino acid substitutions. Inanother embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 4 conservativeamino acid substitutions. In another embodiment, the CTP peptide is avariant of chorionic gonadotrophin CTP which differs from the native CTPby 5 conservative amino acid substitutions. In another embodiment, theCTP peptide amino acid sequence of the present invention is at least 70%homologous to the native CTP amino acid sequence or a peptide thereof.In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 80% homologous to the native CTP aminoacid sequence or a peptide thereof. In another embodiment, the CTPpeptide amino acid sequence of the present invention is at least 90%homologous to the native CTP amino acid sequence or a peptide thereof.In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 95% homologous to the native CTP aminoacid sequence or a peptide thereof.

In another embodiment, the CTP peptide DNA sequence of the presentinvention is at least 70% homologous to the native CTP DNA sequence or apeptide thereof. In another embodiment, the CTP peptide DNA sequence ofthe present invention is at least 80% homologous to the native CTP DNAsequence or a peptide thereof. In another embodiment, the CTP peptideDNA sequence of the present invention is at least 90% homologous to thenative CTP DNA sequence or a peptide thereof. In another embodiment, theCTP peptide DNA sequence of the present invention is at least 95%homologous to the native CTP DNA sequence or a peptide thereof.

In one embodiment, at least one of the chorionic gonadotrophin CTP aminoacid sequences is truncated. In another embodiment, both of thechorionic gonadotrophin CTP amino acid sequences are truncated. Inanother embodiment, 2 of the chorionic gonadotrophin CTP amino acidsequences are truncated. In another embodiment, 2 or more of thechorionic gonadotrophin CTP amino acid sequences are truncated. Inanother embodiment, all of the chorionic gonadotrophin CTP amino acidsequences are truncated. In one embodiment, the truncated CTP comprisesthe first 10 amino acids of SEQ ID NO:43. In one embodiment, thetruncated CTP comprises the first 11 amino acids of SEQ ID NO:43. In oneembodiment, the truncated CTP comprises the first 12 amino acids of SEQID NO:43. In one embodiment, the truncated CTP comprises the first 13amino acids of SEQ ID NO:43. In one embodiment, the truncated CTPcomprises the first 14 amino acids of SEQ ID NO:43. In one embodiment,the truncated CTP comprises the first 15 amino acids of SEQ ID NO:43. Inone embodiment, the truncated CTP comprises the first 16 amino acids ofSEQ ID NO:43. In one embodiment, the truncated CTP comprises the last 14amino acids of SEQ ID NO:43.

In one embodiment, at least one of the chorionic gonadotrophin CTP aminoacid sequences is glycosylated. In another embodiment, both of thechorionic gonadotrophin CTP amino acid sequences are glycosylated. Inanother embodiment, 2 of the chorionic gonadotrophin CTP amino acidsequences are glycosylated. In another embodiment, 2 or more of thechorionic gonadotrophin CTP amino acid sequences are glycosylated. Inanother embodiment, all of the chorionic gonadotrophin CTP amino acidsequences are glycosylated. In one embodiment, the CTP sequence of thepresent invention comprises at least one glycosylation site. In oneembodiment, the CTP sequence of the present invention comprises 2glycosylation sites. In one embodiment, the CTP sequence of the presentinvention comprises 3 glycosylation sites. In one embodiment, the CTPsequence of the present invention comprises 4 glycosylation sites. Eachpossibility represents a separate embodiment of the present invention.

As provided herein, attachment of CTP sequence to both the amino andcarboxy termini of the EPO protein results in increased potency atstimulating erythropoiesis (FIGS. 3-5) and (Table 6 of Example 4), ascompared to recombinant EPO and other combinations of EPO and CTP. Insome embodiments, an EPO attached to three CTP sequences does not impairbinding to its receptor as evidenced in Table 4 of Example 3, whichdemonstrates that EPO attached to three CTP sequences is equallyeffective at stimulating proliferation of TF-1 cells as wild-type EPO.

In some embodiments, “homology” according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution, thereof and biologically active polypeptidefragments thereof. In one embodiment the substitution variant comprisesa glycine in position 104 of erythropoietin amino acid sequence issubstituted by a serine (SEQ ID NO: 22).

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 1 having additionally at least one CTP amino acid peptide on theN-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 1 having additionally at least one CTP amino acid peptide on theN-terminus and at least one additional CTP amino acid peptide on theC-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 2 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 3having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 4having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 5having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 6having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 16having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 22having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment of anemia. Inanother embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for the treatment of anemia. In another embodiment,the methods of the present invention provide a nucleic acid set forth inSEQ ID NO: 20 encoding an EPO peptide and one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of anemia. In another embodiment, the methods of thepresent invention provide a nucleic acid set forth in SEQ ID NO: 21encoding an EPO peptide and one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for the treatment ofanemia.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor inhibiting anemia. In another embodiment, the methods of the presentinvention provide an EPO peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for inhibiting anemia. In another embodiment, the methods ofthe present invention provide an EPO peptide set forth in SEQ ID NO: 1having additionally at least one CTP amino acid peptide on theN-terminus for inhibiting anemia. In another embodiment, the methods ofthe present invention provide an EPO peptide set forth in SEQ ID NO: 1having additionally at least one CTP amino acid peptide on theN-terminus and at least additional one CTP amino acid peptide on theC-terminus for inhibiting anemia. In another embodiment, the methods ofthe present invention provide an EPO peptide set forth in SEQ ID NO: 2having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting anemia. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 3 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for inhibitinganemia. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 4 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for inhibiting anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 5 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 6 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting anemia. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 16 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for inhibitinganemia. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 22 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for inhibiting anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for inhibiting anemia. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding an EPO peptide having one CTP amino acid peptide onthe N-terminus and two CTP amino acid peptides on the C-terminus forinhibiting anemia. In another embodiment, the methods of the presentinvention provide a nucleic acid set forth in SEQ ID NO: 20 encoding anEPO peptide and one CTP amino acid peptide on the N-terminus and atleast one CTP amino acid peptide on the C-terminus for inhibitinganemia. In another embodiment, the methods of the present inventionprovide a nucleic acid set forth in SEQ ID NO: 21 encoding an EPOpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for inhibiting anemia.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for the treatment oftumor-associated anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 1having additionally at least one CTP amino acid peptide on theN-terminus for the treatment of tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 1 having additionally at least one CTP aminoacid peptide on the N-terminus and at least additional one CTP aminoacid peptide on the C-terminus for the treatment of tumor-associatedanemia. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 2 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment oftumor-associated anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 3having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 4 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 5 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 6 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 16 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 22 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of tumor-associated anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment oftumor-associated anemia. In another embodiment, the methods of thepresent invention provide a nucleic acid sequence encoding an EPOpeptide having additionally one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for the treatment oftumor-associated anemia. In another embodiment, the methods of thepresent invention provide a nucleic acid set forth in SEQ ID NO: 20encoding an EPO peptide having additionally one CTP amino acid peptideon the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of tumor-associated anemia. In anotherembodiment, the methods of the present invention provide a nucleic acidset forth in SEQ ID NO: 21 encoding an EPO peptide having additionallyone CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for the treatment of tumor-associated anemia.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor inhibiting tumor-associated anemia. In another embodiment, themethods of the present invention provide an EPO peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for inhibiting tumor-associatedanemia. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 1 having additionally atleast one CTP amino acid peptide on the N-terminus for inhibitingtumor-associated anemia. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 1having additionally at least one CTP amino acid peptide on theN-terminus and at least additional one CTP amino acid peptide on theC-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 2 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 3 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 4 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 5 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 6 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 16 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 22 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting tumor-associated anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for inhibiting tumor-associatedanemia. In another embodiment, the methods of the present inventionprovide a nucleic acid sequence encoding an EPO peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for inhibiting tumor-associatedanemia. In another embodiment, the methods of the present inventionprovide a nucleic acid set forth in SEQ ID NO: 20 encoding an EPOpeptide and one CTP amino acid peptide on the N-terminus and at leastone CTP amino acid peptide on the C-terminus for inhibitingtumor-associated anemia. In another embodiment, the methods of thepresent invention provide a nucleic acid set forth in SEQ ID NO: 21encoding an EPO peptide and one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for inhibitingtumor-associated anemia.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of tumor hypoxia. In another embodiment, the methodsof the present invention provide an EPO peptide having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 1 having additionally at least one CTP aminoacid peptide on the N-terminus for the treatment of tumor hypoxia. Inanother embodiment, the methods of the present invention provide an EPOpeptide set forth in SEQ ID NO: 1 having additionally at least one CTPamino acid peptide on the N-terminus and at least additional one CTPamino acid peptide on the C-terminus for the treatment of tumor hypoxia.In another embodiment, the methods of the present invention provide anEPO peptide set forth in SEQ ID NO: 2 having additionally at least oneCTP amino acid peptide on the N-terminus and at least one CTP amino acidpeptide on the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 3 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 4 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 5 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 6 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 16 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 22 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment of tumor hypoxia.In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide a nucleic acidset forth in SEQ ID NO: 20 encoding an EPO peptide and one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of tumor hypoxia. In anotherembodiment, the methods of the present invention provide a nucleic acidset forth in SEQ ID NO: 21 encoding an EPO peptide having additionallyone CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for the treatment of tumor hypoxia.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for the treatment of chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 1 having additionally at least one CTP amino acid peptide onthe N-terminus for the treatment of chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 1 having additionally at least one CTP amino acid peptide onthe N-terminus and at least additional one CTP amino acid peptide on theC-terminus for the treatment of chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 2 having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 3having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 4having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 5having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 6having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 16having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 22having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment of chronicinfections such as HIV, inflammatory bowel disease, or septic episodes.In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for the treatment of chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide a nucleic acid set forth inSEQ ID NO: 20 encoding an EPO peptide and one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide a nucleic acid set forth in SEQ ID NO: 21encoding an EPO peptide and one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for the treatment ofchronic infections such as HIV, inflammatory bowel disease, or septicepisodes.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor inhibiting chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for inhibiting chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 1 having additionally at least one CTP amino acid peptide onthe N-terminus for inhibiting chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 1 having additionally at least one CTP amino acid peptide onthe N-terminus and at least additional one CTP amino acid peptide on theC-terminus for inhibiting chronic infections such as HIV, inflammatorybowel disease, or septic episodes. In another embodiment, the methods ofthe present invention provide an EPO peptide set forth in SEQ ID NO: 2having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting chronic infections such as HIV, inflammatory bowel disease,or septic episodes. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 3 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for inhibitingchronic infections such as HIV, inflammatory bowel disease, or septicepisodes. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 4 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for inhibiting chronic infectionssuch as HIV, inflammatory bowel disease, or septic episodes. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 5 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for inhibiting chronic infections such as HIV,inflammatory bowel disease, or septic episodes. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 6 having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor inhibiting chronic infections such as HIV, inflammatory boweldisease, or septic episodes. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 16having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting chronic infections such as HIV, inflammatory bowel disease,or septic episodes. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 22 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for inhibitingchronic infections such as HIV, inflammatory bowel disease, or septicepisodes.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for inhibiting chronic infectionssuch as HIV, inflammatory bowel disease, or septic episodes. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding an EPO peptide having one CTP amino acid peptide onthe N-terminus and two CTP amino acid peptides on the C-terminus forinhibiting chronic infections such as HIV, inflammatory bowel disease,or septic episodes. In another embodiment, the methods of the presentinvention provide a nucleic acid set forth in SEQ ID NO: 20 encoding anEPO peptide and one CTP amino acid peptide on the N-terminus and atleast one CTP amino acid peptide on the C-terminus for inhibitingchronic infections such as HIV, inflammatory bowel disease, or septicepisodes. In another embodiment, the methods of the present inventionprovide a nucleic acid set forth in SEQ ID NO: 21 encoding an EPOpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for inhibiting chronic infectionssuch as HIV, inflammatory bowel disease, or septic episodes.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of fatigue syndrome following cancer chemotherapy. Inanother embodiment, the methods of the present invention provide an EPOpeptide having additionally one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for the treatment offatigue syndrome following cancer chemotherapy. In another embodiment,the methods of the present invention provide an EPO peptide set forth inSEQ ID NO: 1 having additionally at least one CTP amino acid peptide onthe N-terminus for the treatment of fatigue syndrome following cancerchemotherapy. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 1 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least additional one CTP amino acid peptide on the C-terminus for thetreatment of fatigue syndrome following cancer chemotherapy. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 2 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of fatigue syndrome following cancerchemotherapy. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 3 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for the treatmentof fatigue syndrome following cancer chemotherapy. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 4 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of fatigue syndrome following cancerchemotherapy. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 5 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for the treatmentof fatigue syndrome following cancer chemotherapy. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 6 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of fatigue syndrome following cancerchemotherapy. In another embodiment, the methods of the presentinvention provide an EPO peptide set forth in SEQ ID NO: 16 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for the treatmentof fatigue syndrome following cancer chemotherapy. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 22 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of fatigue syndrome following cancerchemotherapy.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment of fatiguesyndrome following cancer chemotherapy. In another embodiment, themethods of the present invention provide a nucleic acid sequenceencoding an EPO peptide having additionally one CTP amino acid peptideon the N-terminus and two CTP amino acid peptides on the C-terminus forthe treatment of fatigue syndrome following cancer chemotherapy. Inanother embodiment, the methods of the present invention provide anucleic acid set forth in SEQ ID NO: 20 encoding an EPO peptide and oneCTP amino acid peptide on the N-terminus and at least one CTP amino acidpeptide on the C-terminus for the treatment of fatigue syndromefollowing cancer chemotherapy. In another embodiment, the methods of thepresent invention provide a nucleic acid set forth in SEQ ID NO: 21encoding an EPO peptide and one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for the treatment offatigue syndrome following cancer chemotherapy.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor improving stem cell engraftment. In another embodiment, the methodsof the present invention provide an EPO peptide having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for improving stem cell engraftment. In anotherembodiment, the methods of the present invention provide an EPO peptideset forth in SEQ ID NO: 1 having additionally at least one CTP aminoacid peptide on the N-terminus for improving stem cell engraftment. Inanother embodiment, the methods of the present invention provide an EPOpeptide set forth in SEQ ID NO: 1 having additionally at least one CTPamino acid peptide on the N-terminus and at least additional one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 2 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 3 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 4 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 5 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 6 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 16 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide an EPO peptide set forth in SEQ ID NO: 22 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide a nucleic acid sequence encoding an EPO peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide a nucleic acid set forth in SEQ ID NO: 20 encoding an EPOpeptide and one CTP amino acid peptide on the N-terminus and at leastone CTP amino acid peptide on the C-terminus for improving stem cellengraftment. In another embodiment, the methods of the present inventionprovide a nucleic acid set forth in SEQ ID NO: 21 encoding an EPOpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for improving stem cellengraftment.

In another embodiment, the methods of the present invention provide anEPO peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor increasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for increasing the survival rate of a patient withaplastic anemia or myelodysplastic syndrome. In another embodiment, themethods of the present invention provide an EPO peptide set forth in SEQID NO: 1 having additionally at least one CTP amino acid peptide on theN-terminus for increasing the survival rate of a patient with aplasticanemia or myelodysplastic syndrome. In another embodiment, the methodsof the present invention provide an EPO peptide set forth in SEQ ID NO:1 having additionally at least one CTP amino acid peptide on theN-terminus and at least additional one CTP amino acid peptide on theC-terminus for increasing the survival rate of a patient with aplasticanemia or myelodysplastic syndrome. In another embodiment, the methodsof the present invention provide an EPO peptide set forth in SEQ ID NO:2 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 3having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 4having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 5having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 6having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 16having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide an EPO peptide set forth in SEQ ID NO: 22having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO peptide having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for increasing the survival rate ofa patient with aplastic anemia or myelodysplastic syndrome. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding an EPO peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for increasing the survival rate of a patient with aplasticanemia or myelodysplastic syndrome. In another embodiment, the methodsof the present invention provide a nucleic acid set forth in SEQ ID NO:20 encoding an EPO peptide and one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide a nucleic acid set forth in SEQ ID NO: 21encoding an EPO peptide and one CTP amino acid peptide on the N-terminusand two CTP amino acid peptides on the C-terminus for increasing thesurvival rate of a patient with aplastic anemia or myelodysplasticsyndrome.

In some embodiments, human growth hormone (hGH) is utilized according tothe teachings of the present invention. In some embodiments, theattachment of CTP sequence to both the amino and carboxy termini of thehGH protein results in increased potency (FIG. 11). In some embodiments,the attachment of CTP sequence to both the amino and carboxy termini ofthe hGH protein results in prolonged in-vivo activity. In oneembodiment, CTP-hGH polypeptides of the present invention are set forthin SEQ ID NO: 39-41.

As provided herein, growth gain was demonstrated in hipophysectomizedrats (which have no growth hormone secretion) following injections ofCTP-hGH.

In one embodiment, the phrase “human growth hormone” (hGH) refers to apolypeptide, such as set forth in Genbank Accession No. P01241 (SEQ IDNO: 47), exhibiting hGH activity (i.e. stimulation of growth).

In another embodiment, “human growth hormone” (hGH) refers to apolypeptide, such as set forth in Genbank Accession No. P01241,exhibiting hGH activity (i.e. stimulation of growth).

In another embodiment, “GH” of the present invention also refers tohomologues. In another embodiment, a GH amino acid sequence of methodsand compositions the present invention is at least 50% homologous to aGH sequence set forth herein as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In another embodiment, the percent homology is 60%. Inanother embodiment, the percent homology is 70%. In another embodiment,the percent homology is 80%. In another embodiment, the percent homologyis 90%. In another embodiment, the percent homology is at least 95%. Inanother embodiment, the percent homology is greater than 95%. Eachpossibility represents a separate embodiment of the present invention.

Exemplary CTP-GH polypeptides and CTP-hGH polypeptides of the presentinvention are set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO:41.

In another embodiment, the methods of the present invention provide agrowth hormone (GH) peptide having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for stimulating muscle growth. In another embodiment,the methods of the present invention provide a GH peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for stimulating muscle growth. Inanother embodiment, the methods of the present invention provide a GHpeptide set forth in SEQ ID NO: 23 having additionally at least one CTPamino acid peptide on the N-terminus and at least one CTP amino acidpeptide on the C-terminus for stimulating muscle growth. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 36 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for stimulating muscle growth. In another embodiment,the methods of the present invention provide a GH peptide set forth inSEQ ID NO: 37 having additionally at least one CTP amino acid peptide onthe N-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 38, having additionally at least one CTP amino acid peptide onthe N-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 39 for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 40 for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 41 for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 42 having additionally at least one CTP amino acid peptide on theN-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 44 for stimulating muscle growth.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for stimulating muscle growth. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for stimulating muscle growth. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forstimulating muscle growth. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for stimulating muscle growth.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forstimulating bone growth. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for stimulating bone growth. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forstimulating bone growth. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for stimulatingbone growth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for stimulating bonegrowth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for stimulating bonegrowth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for stimulating bonegrowth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 40 for stimulating bonegrowth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 41 for stimulating bonegrowth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 42 having additionally atleast one CTP amino acid peptide on the N-terminus for stimulating bonegrowth. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 44 for stimulating bonegrowth.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for stimulating bone growth. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for stimulating bone growth. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forstimulating bone growth. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for stimulating bone growth.

In another embodiment, the methods of the present invention provide a GHpeptide of the present invention for maintaining muscle quality.

In another embodiment, the methods of the present invention provide a GHof the present invention for maintaining bone quality.

In another embodiment, the methods of the present invention provide aGH-CTP nucleic acid sequence of the present invention for maintainingbone quality.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for treating a wasting disease. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for treating awasting disease. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 44 fortreating a wasting disease.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for treating a wasting disease. Inanother embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for treating a wasting disease. In another embodiment,the methods of the present invention provide a nucleic acid of SEQ IDNO: 45 encoding a GH peptide comprising one CTP amino acid peptide onthe N-terminus and two CTP amino acid peptides on the C-terminus fortreating a wasting disease. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for treating a wasting disease.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for increasing cardiac function. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for increasingcardiac function. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 44 forincreasing cardiac function.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for increasing cardiac function. Inanother embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for increasing cardiac function. In another embodiment,the methods of the present invention provide a nucleic acid of SEQ IDNO: 45 encoding a GH peptide comprising one CTP amino acid peptide onthe N-terminus and two CTP amino acid peptides on the C-terminus forincreasing cardiac function. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for increasing cardiac function.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing lipolysis. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for increasing lipolysis. In another embodiment, the methodsof the present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forincreasing lipolysis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 40 for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 41 for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 42 having additionally atleast one CTP amino acid peptide on the N-terminus for increasinglipolysis. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 44 for increasinglipolysis.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for increasing lipolysis. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for increasing lipolysis. In another embodiment, the methodsof the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forincreasing lipolysis. In another embodiment, the methods of the presentinvention provide a nucleic acid of SEQ ID NO: 46 encoding a GH peptideand one CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for increasing lipolysis.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving fluid balance. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for improvingfluid balance. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 44 forimproving fluid balance.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for improving fluid balance. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving fluid balance. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forimproving fluid balance. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for improving fluid balance.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for treating osteoporosis. In another embodiment, the methodsof the present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 39 for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 40 for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 41 for treatingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 44 for treatingosteoporosis.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for treating osteoporosis. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for treating osteoporosis. In another embodiment, the methodsof the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus fortreating osteoporosis. In another embodiment, the methods of the presentinvention provide a nucleic acid of SEQ ID NO: 46 encoding a GH peptideand one CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for treating osteoporosis.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for inhibiting osteoporosis. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for inhibitingosteoporosis. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 44 forinhibiting osteoporosis.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for inhibiting osteoporosis. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for inhibiting osteoporosis. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forinhibiting osteoporosis. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for inhibiting osteoporosis.

In another embodiment, the methods of the present invention provide a GHpeptide of the present invention for improving exercise capacity.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving lung function. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving lung function. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving lung function. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 40 for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 41 for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 42 having additionally atleast one CTP amino acid peptide on the N-terminus for improving lungfunction. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 44 for improving lungfunction.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for improving lung function. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving lung function. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forimproving lung function. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for improving lung function.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving immunity. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving immunity. In another embodiment, the methods ofthe present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forimproving immunity. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for improvingimmunity. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for improvingimmunity. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for improvingimmunity. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for improving immunity.In another embodiment, the methods of the present invention provide a GHpeptide set forth in SEQ ID NO: 40 for improving immunity. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 41 for improving immunity. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 42 having additionally at least one CTP aminoacid peptide on the N-terminus for improving immunity. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 44 for improving immunity.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for improving immunity. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving immunity. In another embodiment, the methods ofthe present invention provide a nucleic acid of SEQ ID NO: 45 encoding aGH peptide comprising one CTP amino acid peptide on the N-terminus andtwo CTP amino acid peptides on the C-terminus for improving immunity. Inanother embodiment, the methods of the present invention provide anucleic acid of SEQ ID NO: 46 encoding a GH peptide and one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for improving immunity.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide having additionally one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for regrowing vital organs. In another embodiment, themethods of the present invention provide a GH peptide set forth in SEQID NO: 23 having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for regrowingvital organs. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 37 havingadditionally at least one CTP amino acid peptide on the N-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 38 havingadditionally at least one CTP amino acid peptide on the N-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 39 forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 40 forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 41 forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 42 havingadditionally at least one CTP amino acid peptide on the N-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a GH peptide set forth in SEQ ID NO: 44 forregrowing vital organs.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for regrowing vital organs. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for regrowing vital organs. In another embodiment, themethods of the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forregrowing vital organs. In another embodiment, the methods of thepresent invention provide a nucleic acid of SEQ ID NO: 46 encoding a GHpeptide and one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for regrowing vital organs.

In another embodiment, the methods of the present invention provide a GHpeptide of the present invention for increasing sense of well-being.

In another embodiment, the methods of the present invention provide a GHpeptide having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forrestoring REM sleep. In another embodiment, the methods of the presentinvention provide a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for restoring REM sleep. In another embodiment, the methodsof the present invention provide a GH peptide set forth in SEQ ID NO: 23having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forrestoring REM sleep. In another embodiment, the methods of the presentinvention provide a GH peptide set forth in SEQ ID NO: 36 havingadditionally at least one CTP amino acid peptide on the N-terminus andat least one CTP amino acid peptide on the C-terminus for restoring REMsleep. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 37 having additionally atleast one CTP amino acid peptide on the N-terminus for restoring REMsleep. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 38 having additionally atleast one CTP amino acid peptide on the N-terminus for restoring REMsleep. In another embodiment, the methods of the present inventionprovide a GH peptide set forth in SEQ ID NO: 39 for restoring REM sleep.In another embodiment, the methods of the present invention provide a GHpeptide set forth in SEQ ID NO: 40 for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 41 for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 42 having additionally at least one CTP aminoacid peptide on the N-terminus for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a GH peptideset forth in SEQ ID NO: 44 for restoring REM sleep.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding a GH peptide having additionally at leastone CTP amino acid peptide on the N-terminus and at least one CTP aminoacid peptide on the C-terminus for restoring REM sleep. In anotherembodiment, the methods of the present invention provide a nucleic acidsequence encoding a GH peptide having additionally one CTP amino acidpeptide on the N-terminus and two CTP amino acid peptides on theC-terminus for restoring REM sleep. In another embodiment, the methodsof the present invention provide a nucleic acid of SEQ ID NO: 45encoding a GH peptide comprising one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forrestoring REM sleep. In another embodiment, the methods of the presentinvention provide a nucleic acid of SEQ ID NO: 46 encoding a GH peptideand one CTP amino acid peptide on the N-terminus and two CTP amino acidpeptides on the C-terminus for restoring REM sleep.

In some embodiments, homology according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution, thereof and biologically active polypeptidefragments thereof. In one embodiment the substitution variant is one, inwhich the glutamine in position 65 of hGH is substituted by a valine(SEQ ID NO: 23) [Gellerfors et al., J Pharm Biomed Anal 1989, 7:173-83].

In some embodiments, interferon is utilized according to the teachingsof the present invention. In some embodiments, the attachment of CTPsequence to both the amino and carboxy termini of the interferon proteinresults in increased potency. In some embodiments, the attachment of CTPsequence to both the amino and carboxy termini of the interferon proteinresults in prolonged in-vivo activity.

In one embodiment, “interferon” refers to the mammalian interferonpolypeptide Type I. In one embodiment, “interferon” refers to themammalian interferon polypeptide Type II. In some embodiments,additional suitable interferon polypeptides as known to those ofordinary skill in the art are utilized. In some embodiments, theinterferon is alpha-interferon. In some embodiments, the interferon isbeta-interferon. In some embodiments, the interferon isgamma-interferon. In some embodiments, the interferon isomega-interferon. In some embodiments, the interferon is a subspeciesinterferon. In one embodiment, the subspecies interferon (IFN) isIFN-α2a. In one embodiment, the subspecies interferon IFN) is IFN-α2b.In one embodiment, the subspecies interferon (IFN) is IFN-β1a. In oneembodiment, the interferon (IFN) subspecies is IFN-β1b.

In one embodiment, interferon of the present invention exhibitsinterferon activity, such as antiviral or antiproliferative activity. Insome embodiments, GenBank accession nos. of non-limiting examples ofinterferons are listed in Table 1 below.

In one embodiment, an interferon of the present invention also refers tohomologues. In one embodiment, interferon amino acid sequence of thepresent invention is at least 50% homologous to an interferon sequencedisclosed herein). In one embodiment, interferon amino acid sequence ofthe present invention is at least 60% homologous an interferon sequencedisclosed herein). In one embodiment, interferon amino acid sequence ofthe present invention is at least 70% homologous an interferon sequencedisclosed herein). In one embodiment, interferon amino acid sequence ofthe present invention is at least 80% homologous to an interferonsequence disclosed herein). In one embodiment, interferon amino acidsequence of the present invention is at least 90% homologous to aninterferon sequence disclosed herein). In one embodiment, interferonamino acid sequence of the present invention is at least 95% homologousan interferon sequence disclosed herein). In some embodiments, homologyaccording to the present invention also encompasses deletions,insertions, or substitution variants, including an amino acidsubstitution, thereof and biologically active polypeptide fragmentsthereof. In one embodiment the cysteine in position 17 of interferon βis substituted by a Serine (SEQ ID NO: 24).

Table 1 below lists examples of interferons with their respective NCBIsequence numbers

TABLE 1 Interferon name NCBI sequence number interferon, α 1 NP_076918.1interferon, α 10 NP_002162.1 interferon, α13 NP_008831.2 interferon, α14NP_002163.1 interferon, α16 NP_002164.1 interferon, α17 NP_067091.1interferon, α2 NP_000596.2 interferon, α21 NP_002166.1 interferon, α4NP_066546.1 interferon, α5 NP_002160.1 interferon, α6 NP_066282.1interferon, α7 NP_066401.2 interferon, α8 NP_002161.2 interferon, β 1NP_002167.1 interferon, ε1 NP_795372.1 interferon, γ NP_000610.2interferon, ε NP_064509.1 interferon, Ω1 NP_002168.1

In another embodiment, the methods of the present invention provide aninterferon beta 1 peptide having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for treating or inhibiting multiple sclerosis. Inanother embodiment, the methods of the present invention provide aninterferon beta 1 peptide having additionally one CTP amino acid peptideon the N-terminus and two CTP amino acid peptides on the C-terminus fortreating or inhibiting multiple sclerosis. In another embodiment, themethods of the present invention provide an interferon beta 1 peptideset forth in SEQ ID NO: 24 having additionally at least one CTP aminoacid peptide on the N-terminus and one CTP amino acid peptide on theC-terminus for treating or inhibiting multiple sclerosis. In anotherembodiment, the methods of the present invention provide an interferonbeta 1 peptide set forth in SEQ ID NO: 24 having additionally on theN-terminus the signal peptide of SEQ ID NO: 26 and at least one CTPamino acid peptide on the N-terminus of SEQ ID NO: 26 and at least oneCTP amino acid peptide on the C-terminus of SEQ ID NO: 24 for treatingor inhibiting multiple sclerosis.

In some embodiments, glucagon-like peptide-1 is utilized according tothe teachings of the present invention. In some embodiments, theattachment of CTP sequences to both the amino and carboxy termini of a“glucagon-like peptide-1” results in increased potency. In someembodiments, the attachment of CTP to both the amino and carboxy terminiof a peptide results in prolonged in-vivo activity. In some embodiments,the attachment of CTP to both the amino and carboxy termini of theglucagon-like peptide-results in prolonged in-vivo activity.

In one embodiment, “glucagon-like peptide-1” (GLP-1) refers to amammalian polypeptide. In one embodiment, “glucagon-like peptide-1”(GLP-1) refers to a human polypeptide. In some embodiments, GLP-1 iscleaved from the glucagon preproprotein (Genbank ID No. NP002045) thathas the ability to bind to the GLP-1 receptor and initiate a signaltransduction pathway resulting in insulinotropic activity. In oneembodiment, “insulinotropic activity” refers to the ability to stimulateinsulin secretion in response to elevated glucose levels, therebycausing glucose uptake by cells and decreased plasma glucose levels. Insome embodiments, GLP-1 polypeptides include, but are not limited tothose described in U.S. Pat. No. 5,118,666; which is incorporated byreference herein.

In one embodiment, “GLP-1” refers to a polypeptide, such as set forth insequences set forth in SEQ ID NO: 25 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). In one embodiment, a GLP-1 of the present invention alsorefers to a GLP-1 homologue. In one embodiment, GLP-1 amino acidsequence of the present invention is at least 50% homologous to GLP-1sequences set forth in SEQ ID NO: 25 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). In one embodiment, GLP-1 amino acid sequence of the presentinvention is at least 60% homologous to GLP-1 sequences set forth in SEQID NO: 25 as determined using BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters). In oneembodiment, GLP-1 amino acid sequence of the present invention is atleast 70% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters). In one embodiment, GLP-1amino acid sequence of the present invention is at least 80% homologousto GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters). In one embodiment, GLP-1 amino acid sequenceof the present invention is at least 90% homologous to GLP-1 sequencesset forth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters). In one embodiment, GLP-1 amino acid sequence of the presentinvention is at least 95% homologous to GLP-1 sequences set forth in SEQID NO: 25 as determined using BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters).

In another embodiment, the methods of the present invention provide aGLP-1 peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor treating or inhibiting type II diabetes. In another embodiment, themethods of the present invention provide a GLP-1 peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for treating or inhibiting type IIdiabetes. In another embodiment, the methods of the present inventionprovide a GLP-1 peptide set forth in SEQ ID NO: 25 having additionallyat least one CTP amino acid peptide on the N-terminus and at least oneCTP amino acid peptide on the C-terminus for treating or inhibiting typeII diabetes.

In one embodiment, the homologue also refers to a deletion, insertion,or substitution variant, including an amino acid substitution, thereofand biologically active polypeptide fragments thereof.

In one embodiment the polypeptide sequence-of-interest is an EPO. In oneembodiment the polypeptide sequence-of-interest is an interferon. Inanother embodiment the polypeptide sequence-of-interest is an hGH. Inanother embodiment the polypeptide sequence-of-interest is a GLP-1. Inanother embodiment the polypeptide sequence-of-interest is an insulin.In another embodiment the polypeptide sequence-of-interest isenkephalin. In another embodiment the polypeptide sequence-of-interestis an ACTH. In another embodiment the polypeptide sequence-of-interestis a glucagon. In another embodiment the polypeptidesequence-of-interest is an insulin-like growth factor. In anotherembodiment the polypeptide sequence-of-interest is an epidermal growthfactor. In another embodiment the polypeptide sequence-of-interest is anacidic or basic fibroblast growth factor. In another embodiment thepolypeptide sequence-of-interest is a platelet-derived growth factor. Inanother embodiment the polypeptide sequence-of-interest is agranulocyte-CSF. In another embodiment the polypeptidesequence-of-interest is a macrophage-CSF. In another embodiment thepolypeptide sequence-of-interest is an IL-2. In another embodiment thepolypeptide sequence-of-interest is an IL-3. In another embodiment thepolypeptide sequence-of-interest is a tumor necrosis factor. In anotherembodiment the polypeptide sequence-of-interest is an LHRH. In anotherembodiment the polypeptide sequence-of-interest is an LHRH analog. Inanother embodiment the polypeptide sequence-of-interest is asomatostatin. In another embodiment the polypeptide sequence-of-interestis a growth hormone releasing factor. In another embodiment thepolypeptide sequence-of-interest is an endorphin. In another embodimentthe polypeptide sequence-of-interest is an alveolar surfactant protein.In another embodiment the polypeptide sequence-of-interest is anatriuretic factor. In another embodiment the polypeptidesequence-of-interest is an adhesion. In another embodiment thepolypeptide sequence-of-interest is an angiostatin. In anotherembodiment the polypeptide sequence-of-interest is an endostatin. Inanother embodiment the polypeptide sequence-of-interest is a receptorpeptide. In another embodiment the polypeptide sequence-of-interest is areceptor binding ligand. In another embodiment the polypeptidesequence-of-interest is an antibody. In another embodiment thepolypeptide sequence-of-interest is an antibody fragment. In anotherembodiment the polypeptide sequence-of-interest is a peptide or aprotein including any modified form.

In another embodiment, the peptide of the invention comprises a peptideof interest having additionally at least one CTP amino acid peptide onthe N-terminus and one CTP amino acid peptide on the C-terminus. Inanother embodiment, the peptide of interest having additionally at leastone CTP amino acid peptide on the N-terminus and one CTP amino acidpeptide on the C-terminus comprises a protein selected from thefollowing list: insulin, Albutein/albumin, Activase altiplase/tPA,adenosine deaminase, immune globulin, glucocerebrosidase,Leukine-sargramostim/GM-CSF, G-CSF, Venoglobulin-S/IgG, Proleukinaldesleukin, DNase, factor VIII, Helixate, L-asparaginase, WinRho SDF RhI, Retavase retaplase/tPA, Factor IX, FSH, globulin, fibrin,interleukin-11, becaplermin/PDGF, lepirudin/herudin, TNF, Thymoglobulin,factor VIa, interferon alpha-2a, interferon alfa n-1, interferonalfa-N3, interferon beta-1b, interferon gamma-1b, Interleukin-2, HGH, ormonoclonal antibodies.

In another embodiment, the methods of the present invention provideinsulin having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of diabetes.

In another embodiment, the methods of the present invention providealbumin having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of hypovolemic shock, hemodialysis or cardiopulmonary bypass.

In another embodiment, the methods of the present invention provideActivase-altiplase/tPA having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of acute myocardial infarction, acutemassive pulmonary embolism, or (change throughout) ischemic stroke.

In another embodiment, the methods of the present invention provideadenosine deaminase having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of severe combined immunodeficiencydisease.

In another embodiment, the methods of the present invention provideimmune globulin having additionally at least one CTP amino acid peptideon the N-terminus and one CTP amino acid peptide on the C-terminus forthe treatment of transplant recipients.

In another embodiment, the methods of the present invention provideimmune globulin is a CMV immune globulin. In another embodiment, themethods of the present invention provide glucocerebrosidase havingadditionally at least one CTP amino acid peptide on the N-terminus andone CTP amino acid peptide on the C-terminus for the treatment ofGaucher disease.

In another embodiment, the methods of the present invention provideLeukine-sargramostim/GM-CSF having additionally at least one CTP aminoacid peptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the Stimulation of hematopoietic progenitor cells.

In another embodiment, the methods of the present invention provideG-CSF having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Neutropenia. In another embodiment, the methods of thepresent invention provide Venoglobulin-S/IgG having additionally atleast one CTP amino acid peptide on the N-terminus and one CTP aminoacid peptide on the C-terminus for the treatment of Immunodeficiencydiseases.

In another embodiment, the methods of the present invention provideProleukin-aldesleukin having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of renal carcinoma or metastatic melanoma.

In another embodiment, the methods of the present invention provideDNase having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Cystic fibrosis.

In another embodiment, the methods of the present invention providefactor VIII having additionally at least one CTP amino acid peptide onthe N-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Hemophilia A.

In another embodiment, the methods of the present invention provideHelixate having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Hemophilia A.

In another embodiment, the methods of the present invention provideL-asparaginase having additionally at least one CTP amino acid peptideon the N-terminus and one CTP amino acid peptide on the C-terminus forthe treatment of acute lymphoblastic leukemia.

In another embodiment, the methods of the present invention provideWinRho SDF Rh IgG having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of Rh isoimmunization and immunethrombocytopenic purpura.

In another embodiment, the methods of the present invention provideRetavase retaplase/tPA having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of acute myocardial infarction.

In another embodiment, the methods of the present invention provideFactor IX having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of Hemophilia B.

In another embodiment, the methods of the present invention provideFactor IX having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of Hemophilia B.

In another embodiment, the methods of the present invention provide FSHhaving additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forstimulation of ovulation during assisted reproduction.

In another embodiment, the methods of the present invention provideglobulin having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe prevention of respiratory syncytial virus disease.

In another embodiment, the methods of the present invention providefibrin having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forwound management and hemostasis. In another embodiment, the methods ofthe present invention provide interleukin-11 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for chemotherapy-inducedthrombocytopenia.

In another embodiment, the methods of the present invention providebecaplermin/PDGF having additionally at least one CTP amino acid peptideon the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of diabetic foot ulcers.

In another embodiment, the methods of the present invention providelepirudin/herudin having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for anticoagulation in heparin-induced thrombocytopenia.

In another embodiment, the methods of the present invention providesoluble TNF having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of rheumatoid arthritis.

In another embodiment, the methods of the present invention provideThymoglobulin having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of organ transplant rejection disease.

In another embodiment, the methods of the present invention providefactor VIIa having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of hemophilia.

In another embodiment, the methods of the present invention provideinterferon alpha-2a having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of hairy cell leukemia and AIDS-relatedKaposi's sarcoma.

In another embodiment, the methods of the present invention provideinterferon alpha-2b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of Hairy cell leukemia, genital warts,AIDS-related Kaposi's sarcoma, hepatitis C, hepatitis B, malignantmelanoma, and follicular lymphoma.

In another embodiment, the methods of the present invention provideinterferon alfa-N3 having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of genital warts.

In another embodiment, the methods of the present invention provideinterferon gamma-1b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of chronic granulomatous disease.

In another embodiment, the methods of the present invention provideinterferon alfa n−1 having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of hepatitis C infection.

In another embodiment, the methods of the present invention provideInterleukin-2 having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of renal carcinoma and metastatic melanoma.

In another embodiment, the methods of the present invention provideinterferon beta-1b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of multiple sclerosis.

In another embodiment, the methods of the present invention provide hGHhaving additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of wasting disease, AIDS, cachexia, or hGH deficiency.

In another embodiment, the methods of the present invention provide anOKT3 monoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for organ transplant.

In another embodiment, the methods of the present invention provide aReo monoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for prevention of complications from coronary interventionand angioplasty.

In another embodiment, the methods of the present invention provide amonoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for treating colorectal cancer, Non-Hodgkin's lymphoma,kidney transplant rejection, metastatic breast cancer, or the preventionof respiratory syncytial virus disease.

In one embodiment, the invention is employed in veterinary medicine. Inone embodiment, the present invention provides treatment of domesticatedmammals which are maintained as human companions (e.g., dogs, cats,horses), which have significant commercial value (e.g., dairy cows. beefcattle, sporting animals), which have significant scientific value(e.g., captive or free specimens of endangered species), or whichotherwise have value.

In one embodiment, polypeptides, antibodies, or polynucleotides of thepresent invention are administered to an animal (e.g., mouse, rat,rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat,horse, cow, sheep, dog, cat, non-human primate, and human. In oneembodiment, the recited applications have uses in a wide variety ofhosts. In some embodiments, such hosts include, but are not limited to,human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, ornon-human primate.

In one embodiment, farm animals are treated by the methods of thepresent invention. In one embodiment, farm animals include pigs, cattle,dairy cows, horses, goats, sheep, chickens, turkeys, geese, ducks andrelated species. In one embodiment, laboratory animals are treated bythe methods of the present invention. In one embodiment, laboratoryanimals include rats, mice, guinea pigs, rabbits, goats, monkeys, dogs,cats and others. In one embodiment, zoo animals are treated by themethods of the present invention. In one embodiment, zoo animals includeall vertebrate animals kept in zoos. In one embodiment, aquatic animalsare treated by the methods of the present invention. In one embodiment,aquatic animals include fish, eels, turtles, seals, penguins, sharks,whales, and related species. In one embodiment, domesticated animals aretreated by the methods of the present invention. In one embodiment,domesticated animals include any pet, such as cats and dogs, or animalthat is kept by humans, e.g., horses, cattle, pigs, goats, rabbits,chickens, turkeys, geese, ducks and the like.

According to the present invention the term pigs includes pigs, piglets,hogs, gilts, barrows, boars and sows. In another embodiment, “cattle”refers to calves, cows, dairy cows, heifers, steers and bulls.

In one embodiment, bovine growth hormone is utilized by the methods ofthe present invention. In one embodiment, artificial bovine growthhormone is utilized by the methods of the present invention. In oneembodiment, the artificial bovine growth hormone has a sequence setforth in NCBI sequence ID number AAA72262. In another embodiment, theartificial bovine growth hormone is any other artificial bovine growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, sheep growth hormone is utilized by the methods ofthe present invention. In one embodiment, sheep growth hormone has asequence set forth in NCBI sequence ID number NP_(—)001009315. Inanother embodiment, the sheep growth hormone is any other sheep growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, horse growth hormone is utilized by the methods ofthe present invention. In one embodiment, horse growth hormone has asequence set forth in NCBI sequence ID number AAA21027. In anotherembodiment, the horse growth hormone is any other horse growth hormoneknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, chicken growth hormone is utilized by the methods ofthe present invention. In one embodiment, chicken growth hormone has asequence set forth in NCBI sequence ID number CAA3561. In anotherembodiment, the chicken growth hormone is any other chicken growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, murine growth hormone is utilized by the methods ofthe present invention. In one embodiment, the murine growth hormone hasa sequence set forth in NCBI sequence ID number NP_(—)032143. In anotherembodiment, the murine growth hormone is any other murine growth hormoneknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, tilapia growth hormone is utilized by the methods ofthe present invention. In one embodiment, the tilapia growth hormone hasa sequence set forth in NCBI sequence ID number CAA00818. In anotherembodiment, the tilapia growth hormone is any other tilapia growthhormone known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, bovine EPO is utilized by the methods of the presentinvention. In one embodiment, artificial bovine growth hormone isutilized by the methods of the present invention. In one embodiment,artificial bovine growth hormone has a sequence set forth in NCBIsequence ID number NP_(—)776334. In another embodiment, the bovine EPOis any other bovine EPO known in the art. Each possibility represents aseparate embodiment of the present invention.

In one embodiment, pig EPO is utilized by the methods of the presentinvention. In one embodiment, pig EPO has a sequence set forth in NCBIsequence ID number NP_(—)999299. In another embodiment, the pig EPO isany other pig EPO known in the art. Each possibility represents aseparate embodiment of the present invention.

In one embodiment, sheep EPO is utilized by the methods of the presentinvention. In one embodiment, sheep growth hormone has a sequence setforth in NCBI sequence ID number NP_(—)001019908. In another embodiment,the sheep growth hormone is any other sheep growth hormone known in theart. Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, murine EPO is utilized by the methods of the presentinvention. In one embodiment, the murine growth hormone has a sequenceset forth in NCBI sequence ID number CAA72707. In another embodiment,the murine growth hormone is any other murine growth hormone known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In one embodiment, bovine GLP-1 is utilized by the methods of thepresent invention. In one embodiment, bovine GLP-1 has a sequence setforth in NCBI sequence ID number P01272. In another embodiment, thebovine GLP-1 is any other bovine GLP-1 known in the art. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, sheep GLP-1 is utilized by the methods of the presentinvention. In one embodiment, sheep GLP-1 has a sequence set forth inNCBI sequence ID number Q8MJ25. In another embodiment, the sheep GLP-1is any other sheep GLP-1 known in the art. Each possibility represents aseparate embodiment of the present invention.

In one embodiment, pig GLP-1 is utilized by the methods of the presentinvention. In one embodiment, chicken GLP-1 has a sequence set forth inNCBI sequence ID number P01274. In another embodiment, the chicken GLP-1is any other chicken GLP-1 known in the art. Each possibility representsa separate embodiment of the present invention.

In one embodiment, murine GLP-1 is utilized by the methods of thepresent invention. In one embodiment, the murine GLP-1 has a sequenceset forth in NCBI sequence ID number NP_(—)032127. In anotherembodiment, the murine GLP-1 is any other murine GLP-1 known in the art.Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, bovine interferon alpha is utilized by the methods ofthe present invention. In one embodiment, bovine interferon alpha has asequence set forth in NCBI sequence ID number ABD57311. In anotherembodiment, the bovine interferon alpha is any other bovine interferonalpha known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, sheep interferon alpha is utilized by the methods ofthe present invention. In one embodiment, sheep interferon alpha has asequence set forth in NCBI sequence ID number CAA41790. In anotherembodiment, the sheep interferon alpha is any other sheep interferonalpha known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, pig interferon alpha is utilized by the methods ofthe present invention. In one embodiment, chicken interferon alpha has asequence set forth in NCBI sequence ID number AAP92118. In anotherembodiment, the pig interferon alpha is any other pig interferon alphaknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, murine interferon alpha is utilized by the methods ofthe present invention. In one embodiment, the murine interferon alphahas a sequence set forth in NCBI sequence ID number AAA37886. In anotherembodiment, the murine interferon alpha is any other murine interferonalpha known in the art. Each possibility represents a separateembodiment of the present invention.

In some embodiments, the CTP sequences modification is advantageous inpermitting lower dosages to be used.

In some embodiments, “polypeptide” as used herein encompasses nativepolypeptides (either degradation products, synthetically synthesizedpolypeptides or recombinant polypeptides) and peptidomimetics(typically, synthetically synthesized polypeptides), as well as peptoidsand semipeptoids which are polypeptide analogs, which have, in someembodiments, modifications rendering the polypeptides even more stablewhile in a body or more capable of penetrating into cells.

In some embodiments, modifications include, but are not limited to Nterminus modification, C terminus modification, polypeptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

In some embodiments, polypeptide bonds (—CO—NH—) within the polypeptideare substituted. In some embodiments, the polypeptide bonds aresubstituted by N-methylated bonds (—N(CH3)-CO—). In some embodiments,the polypeptide bonds are substituted by ester bonds(—C(R)H—C—O—O—C(R)—N—). In some embodiments, the polypeptide bonds aresubstituted by ketomethylen bonds (—CO—CH2-). In some embodiments, thepolypeptide bonds are substituted by (x-aza bonds (—NH—N(R)—CO—),wherein R is any alkyl, e.g., methyl, carba bonds (—CH2-NH—). In someembodiments, the polypeptide bonds are substituted by hydroxyethylenebonds (—CH(OH)—CH2-). In some embodiments, the polypeptide bonds aresubstituted by thioamide bonds (—CS—NH—). In some embodiments, thepolypeptide bonds are substituted by olefinic double bonds (—CH═CH—). Insome embodiments, the polypeptide bonds are substituted by retro amidebonds (—NH—CO—). In some embodiments, the polypeptide bonds aresubstituted by polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the“normal” side chain, naturally presented on the carbon atom. In someembodiments, these modifications occur at any of the bonds along thepolypeptide chain and even at several (2-3 bonds) at the same time.

In some embodiments, natural aromatic amino acids of the polypeptidesuch as Trp, Tyr and Phe, be substituted for synthetic non-natural acidsuch as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylatedderivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. Insome embodiments, the polypeptides of the present invention include oneor more modified amino acid or one or more non-amino acid monomers (e.g.fatty acid, complex carbohydrates etc).

In one embodiment, “amino acid” or “amino acid” is understood to includethe 20 naturally occurring amino acid; those amino acid often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment,“amino acid” includes both D- and L-amino acid.

In some embodiments, the polypeptides of the present invention areutilized in therapeutics which requires the polypeptides to be in asoluble form. In some embodiments, the polypeptides of the presentinvention include one or more non-natural or natural polar amino acid,including but not limited to serine and threonine which are capable ofincreasing polypeptide solubility due to their hydroxyl-containing sidechain.

In some embodiments, the polypeptides of the present invention areutilized in a linear form, although it will be appreciated by oneskilled in the art that in cases where cyclicization does not severelyinterfere with polypeptide characteristics, cyclic forms of thepolypeptide can also be utilized.

In some embodiments, the polypeptides of present invention arebiochemically synthesized such as by using standard solid phasetechniques. In some embodiments, these biochemical methods includeexclusive solid phase synthesis, partial solid phase synthesis, fragmentcondensation, or classical solution synthesis. In some embodiments,these methods are used when the polypeptide is relatively short (about5-15 kDa) and/or when it cannot be produced by recombinant techniques(i.e., not encoded by a nucleic acid sequence) and therefore involvesdifferent chemistry.

In some embodiments, solid phase polypeptide synthesis procedures arewell known to one skilled in the art and further described by JohnMorrow Stewart and Janis Dillaha Young, Solid Phase PolypeptideSyntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments,synthetic polypeptides are purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing by methods known to oneskilled in the art.

In some embodiments, recombinant protein techniques are used to generatethe polypeptides of the present invention. In some embodiments,recombinant protein techniques are used for generation of relativelylong polypeptides (e.g., longer than 18-25 amino acid). In someembodiments, recombinant protein techniques are used for the generationof large amounts of the polypeptide of the present invention. In someembodiments, recombinant techniques are described by Bitter et al.,(1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al, (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

In one embodiment, a polypeptide of the present invention is synthesizedusing a polynucleotide encoding a polypeptide of the present invention.In some embodiments, the polynucleotide encoding a polypeptide of thepresent invention is ligated into an expression vector, comprising atranscriptional control of a cis-regulatory sequence (e.g., promotersequence). In some embodiments, the cis-regulatory sequence is suitablefor directing constitutive expression of the polypeptide of the presentinvention. In some embodiments, the cis-regulatory sequence is suitablefor directing tissue specific expression of the polypeptide of thepresent invention. In some embodiments, the cis-regulatory sequence issuitable for directing inducible expression of the polypeptide of thepresent invention.

In some embodiments, polynucleotides which express the polypeptides ofthe present invention are as set forth in SEQ ID NOs: 20, 21, 44, 45 and46.

In some embodiment, tissue-specific promoters suitable for use with thepresent invention include sequences which are functional in specificcell population, example include, but are not limited to promoters suchas albumin that is liver specific [Pinkert et al., (1987) Genes Dev.1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv.Immunol. 43:235-275]; in particular promoters of T-cell receptors[Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerjiet al. (1983) Cell 33729-740], neuron-specific promoters such as theneurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985)Science 230:912-916] or mammary gland-specific promoters such as themilk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Inducible promoters suitable for use with thepresent invention include for example the tetracycline-induciblepromoter (Srour, M. A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase “a polynucleotide” refers to a single ordouble stranded nucleic acid sequence which be isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

In one embodiment, “complementary polynucleotide sequence” refers to asequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.In one embodiment, the sequence can be subsequently amplified in vivo orin vitro using a DNA polymerase.

In one embodiment, “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

In one embodiment, “composite polynucleotide sequence” refers to asequence, which is at least partially complementary and at leastpartially genomic. In one embodiment, a composite sequence can includesome exonal sequences required to encode the polypeptide of the presentinvention, as well as some intronic sequences interposing there between.In one embodiment, the intronic sequences can be of any source,including of other genes, and typically will include conserved splicingsignal sequences. In one embodiment, intronic sequences include cisacting expression regulatory elements.

In one embodiment, the polynucleotides of the present invention furthercomprise a signal sequence encoding a signal peptide for the secretionof the polypeptides of the present invention. In some embodiments,signal sequences include, but are not limited to the endogenous signalsequence for EPO as set forth in SEQ ID NO: 19 or the endogenous signalsequence for IFN-β1 as set forth in SEQ ID NO: 26. In anotherembodiment, the signal sequence is N-terminal to the CTP sequence thatis in turn N-terminal to the polypeptide sequence of interest; e.g. thesequence is (a) signal sequence- (b) CTP- (c) sequence-of-interest- (d)optionally 1 or more additional CTP sequences. In another embodiment, 1or more CTP sequences is inserted between the signal sequence of apolypeptide sequence of interest and the polypeptide sequence ofinterest itself, thus interrupting the wild-type sequence of interest.Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, following expression and secretion, the signalpeptides are cleaved from the precursor proteins resulting in the matureproteins.

In some embodiments, polynucleotides of the present invention areprepared using PCR techniques as described in Example 1, or any othermethod or procedure known to one skilled in the art. In someembodiments, the procedure involves the legation of two different DNAsequences (See, for example, “Current Protocols in Molecular Biology”,eds. Ausubel et al., John Wiley & Sons, 1992).

In one embodiment, polynucleotides of the present invention are insertedinto expression vectors (i.e., a nucleic acid construct) to enableexpression of the recombinant polypeptide. In one embodiment, theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes. In one embodiment, the expression vector of the presentinvention includes additional sequences which render this vectorsuitable for replication and integration in eukaryotes. In oneembodiment, the expression vector of the present invention includes ashuttle vector which renders this vector suitable for replication andintegration in both prokaryotes and eukaryotes. In some embodiments,cloning vectors comprise transcription and translation initiationsequences (e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals).

In one embodiment, a variety of prokaryotic or eukaryotic cells can beused as host-expression systems to express the polypeptides of thepresent invention. In some embodiments, these include, but are notlimited to, microorganisms, such as bacteria transformed with arecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvector containing the polypeptide coding sequence; yeast transformedwith recombinant yeast expression vectors containing the polypeptidecoding sequence; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors,such as Ti plasmid, containing the polypeptide coding sequence.

In some embodiments, non-bacterial expression systems are used (e.g.mammalian expression systems such as CHO cells) to express thepolypeptide of the present invention. In one embodiment, the expressionvector used to express polynucleotides of the present invention inmammalian cells is pCI-DHFR vector comprising a CMV promoter and aneomycin resistance gene. Construction of the pCI-dhfr vector isdescribed, according to one embodiment, in Example 1.

In some embodiments, in bacterial systems of the present invention, anumber of expression vectors can be advantageously selected dependingupon the use intended for the polypeptide expressed. In one embodiment,large quantities of polypeptide are desired. In one embodiment, vectorsthat direct the expression of high levels of the protein product,possibly as a fusion with a hydrophobic signal sequence, which directsthe expressed product into the periplasm of the bacteria or the culturemedium where the protein product is readily purified are desired. In oneembodiment, certain fusion protein engineered with a specific cleavagesite to aid in recovery of the polypeptide. In one embodiment, vectorsadaptable to such manipulation include, but are not limited to, the pETseries of E. coli expression vectors [Studier et al., Methods inEnzymol. 185:60-89 (1990)].

In one embodiment, yeast expression systems are used. In one embodiment,a number of vectors containing constitutive or inducible promoters canbe used in yeast as disclosed in U.S. Pat. No. 5,932,447. In anotherembodiment, vectors which promote integration of foreign DNA sequencesinto the yeast chromosome are used.

In one embodiment, the expression vector of the present invention canfurther include additional polynucleotide sequences that allow, forexample, the translation of several proteins from a single mRNA such asan internal ribosome entry site (IRES) and sequences for genomicintegration of the promoter-chimeric polypeptide.

In some embodiments, mammalian expression vectors include, but are notlimited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB,pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

In some embodiments, expression vectors containing regulatory elementsfrom eukaryotic viruses such as retroviruses are used by the presentinvention. SV40 vectors include pSVT7 and pMT2. In some embodiments,vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Bar virus include pHEBO, and p2O5. Otherexemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

In some embodiments, recombinant viral vectors are useful for in vivoexpression of the polypeptides of the present invention since they offeradvantages such as lateral infection and targeting specificity. In oneembodiment, lateral infection is inherent in the life cycle of, forexample, retrovirus and is the process by which a single infected cellproduces many progeny virions that bud off and infect neighboring cells.In one embodiment, the result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. In one embodiment, viral vectors are produced that are unableto spread laterally. In one embodiment, this characteristic can beuseful if the desired purpose is to introduce a specified gene into onlya localized number of targeted cells.

In one embodiment, various methods can be used to introduce theexpression vector of the present invention into cells. Such methods aregenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992), inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press,Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

In some embodiments, introduction of nucleic acid by viral infectionoffers several advantages over other methods such as lipofection andelectroporation, since higher transfection efficiency can be obtaineddue to the infectious nature of viruses.

In one embodiment, it will be appreciated that the polypeptides of thepresent invention can also be expressed from a nucleic acid constructadministered to the individual employing any suitable mode ofadministration, described hereinabove (i.e., in-vivo gene therapy). Inone embodiment, the nucleic acid construct is introduced into a suitablecell via an appropriate gene delivery vehicle/method (transfection,transduction, homologous recombination, etc.) and an expression systemas needed and then the modified cells are expanded in culture andreturned to the individual (i.e., ex-vivo gene therapy).

In one embodiment, in vivo gene therapy using EPO has been attempted inanimal models such as rodents [Bohl et al., Blood. 2000; 95:2793-2798],primates [Gao et al., Blood, 2004, Volume 103, Number 9] and has provensuccessful in human clinical trials for patients with chronic renalfailure [Lippin et al Blood 2005, 106, Number 7].

In one embodiment, plant expression vectors are used. In one embodiment,the expression of a polypeptide coding sequence is driven by a number ofpromoters. In some embodiments, viral promoters such as the 35S RNA and19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)],or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311(1987)] are used. In another embodiment, plant promoters are used suchas, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] orheat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley etal., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructsare introduced into plant cells using Ti plasmid, Ri plasmid, plantviral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach [Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Otherexpression systems such as insects and mammalian host cell systems,which are well known in the art, can also be used by the presentinvention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the polypeptide), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressedpolypeptide.

Various methods, in some embodiments, can be used to introduce theexpression vector of the present invention into the host cell system. Insome embodiments, such methods are generally described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (1989, 1992), in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Changet al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vegaet al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

In some embodiments, transformed cells are cultured under effectiveconditions, which allow for the expression of high amounts ofrecombinant polypeptide. In some embodiments, effective cultureconditions include, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Inone embodiment, an effective medium refers to any medium in which a cellis cultured to produce the recombinant polypeptide of the presentinvention. In some embodiments, a medium typically includes an aqueoussolution having assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. In some embodiments, cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes and petri plates. In some embodiments,culturing is carried out at a temperature, pH and oxygen contentappropriate for a recombinant cell. In some embodiments, culturingconditions are within the expertise of one of ordinary skill in the art.

In some embodiments, depending on the vector and host system used forproduction, resultant polypeptides of the present invention eitherremain within the recombinant cell, secreted into the fermentationmedium, secreted into a space between two cellular membranes, such asthe periplasmic space in E. coli; or retained on the outer surface of acell or viral membrane.

In one embodiment, following a predetermined time in culture, recoveryof the recombinant polypeptide is effected.

In one embodiment, the phrase “recovering the recombinant polypeptide”used herein refers to collecting the whole fermentation mediumcontaining the polypeptide and need not imply additional steps ofseparation or purification.

In one embodiment, polypeptides of the present invention are purifiedusing a variety of standard protein purification techniques, such as,but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the polypeptide of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the polypeptide can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the polypeptide and the cleavable moiety and the polypeptide canbe released from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

In one embodiment, the polypeptide of the present invention is retrievedin “substantially pure” form.

In one embodiment, the phrase “substantially pure” refers to a puritythat allows for the effective use of the protein in the applicationsdescribed herein.

In one embodiment, the polypeptide of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In one embodiment, production of CTP-EPO-CTP polypeptides usingrecombinant DNA technology is illustrated in Example 1.

In some embodiments, the recombinant polypeptides are synthesized andpurified; their therapeutic efficacy can be assayed either in vivo or invitro. In one embodiment, the binding activities of the recombinant EPOpolypeptides of the present invention can be ascertained using variousassays as described in Examples 2-6 and 8-9. In one embodiment, in vitrobinding activity is ascertained by measuring the ability of thepolypeptide to stimulate proliferation of TF-1 cells. In one embodiment,in vivo activity is deduced by analyzing hematocrit levels (FIGS. 3-5)and/or as a percentage of reticulocytes.

In one embodiment, the EPO polypeptides of the present invention can beused to treat a subject, with a variety of erythropoietin-associatedconditions. In some embodiments, a subject is a human subject.

In some embodiment, the phrase “erythropoietin-associated conditions”refers to any condition associated with below normal, abnormal, orinappropriate modulation of erythropoietin. In some embodiment, levelsof erythropoietin associated with such conditions are determined by anymeasure accepted and utilized by those of skill in the art. In someembodiment, erythropoietin-associated conditions typically includeanemic conditions.

In some embodiment, “anemic conditions” refers to any condition,disease, or disorder associated with anemia. In some embodiment, anemicconditions include, but are not limited to, aplastic anemia, autoimmunehemolytic anemia, bone marrow transplantation, Churg-Strauss syndrome,Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome, graft versushost disease, hematopoietic stem cell transplantation, hemolytic uremicsyndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria,osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpuraSchoenlein-Henoch, sideroblastic anemia, refractory anemia with excessof blasts, rheumatoid arthritis, Shwachman syndrome, sickle celldisease, thalassemia major, thalassemia minor, thrombocytopenic purpura,etc.

In one embodiment, the present invention comprises CTP-hGH-CTPpolypeptides. In one embodiment, recombinant DNA technology methods areused for the production of CTP-hGH-CTP polypeptides as illustrated inExample 7. In one embodiment, the therapeutic efficacy of theCTP-hGH-CTP polypeptides of the present invention is assayed either invivo. In one embodiment, the therapeutic efficacy of the CTP-hGH-CTPpolypeptides of the present invention is assayed either in vitro. In oneembodiment, the binding activities of the recombinant hGH polypeptidesof the present invention are measured using Nb2 (a prolactin-dependentrat lymphoma cell line (ECACC Cell Bank)) or a FCD-P1 murine cell line,previously transfected with human growth hormone receptor. In oneembodiment, binding of hGH to these receptors induces cell proliferationwhich in one embodiment is measured by the levels of MTT cellular stainas a function of hGH activity. In one embodiment, in vivo activity isdeduced by measuring weight gain over time in treated growth hormonedeficient animals.

In some embodiment, human growth hormone polypeptides of the presentinvention can be used to treat a subject, with conditions related togrowth and weight, such as a growth deficiency disorder, AIDS wasting,aging, impaired immune function of HIV-infected subjects, a catabolicillness, surgical recovery, a congestive cardiomyopathy, livertransplantation, liver regeneration after hepatectomy, chronic renalfailure, renal osteodystrophy, osteoporosis,achondroplasia/hypochondroplasia, skeletal dysplasia, a chronicinflammatory or nutritional disorder such as Crohn's disease, shortbowel syndrome, juvenile chronic arthritis, cystic fibrosis, maleinfertility, X-linked hypophosphatemic rickets, Down's syndrome, Spinabifida, Noonan Syndrome, obesity, impaired muscle strength andfibromyalgia. In some embodiments, interferon polypeptides of thepresent invention are used to treat a subject, with a variety ofconditions such as hairy cell leukemia (HCL), Kaposi's sarcoma (KS),chronic myelogenous leukemia (CML), chronic hepatitis C(CHC),condylomata acuminata (CA), chronic hepatitis B, malignant melanoma,follicular non-Hodgkin's lymphoma, multiple sclerosis, chronicgranulomatous disease, Mycobacterium avium complex (MAC), pulmonaryfibrosis and osteoporosis.

In some embodiments, Glucagon-like peptide-1 (GLP-1) polypeptides of thepresent invention are used to treat a subject with non-insulin dependentdiabetes, obesity, stroke, myocardial infarction, stroke, stress-inducedhyperglycemia, or irritable bowel syndrome.

In one embodiment, the polypeptides of the present invention can beprovided to the individual per se. In one embodiment, the polypeptidesof the present invention can be provided to the individual as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

In one embodiment, a “pharmaceutical composition” refers to apreparation of one or more of the active ingredients described hereinwith other chemical components such as physiologically suitable carriersand excipients. The purpose of a pharmaceutical composition is tofacilitate administration of a compound to an organism.

In one embodiment, “active ingredient” refers to the polypeptidesequence of interest, which is accountable for the biological effect.

In some embodiments, any of the compositions of this invention willcomprise at least two CTP sequences bound to a protein of interest, inany form. In one embodiment, the present invention provides combinedpreparations. In one embodiment, “a combined preparation” definesespecially a “kit of parts” in the sense that the combination partnersas defined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partnersi.e., simultaneously, concurrently, separately or sequentially. In someembodiments, the parts of the kit of parts can then, e.g., beadministered simultaneously or chronologically staggered, that is atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partners, in some embodiments, can be administered in thecombined preparation. In one embodiment, the combined preparation can bevaried, e.g., in order to cope with the needs of a patient subpopulationto be treated or the needs of the single patient which different needscan be due to a particular disease, severity of a disease, age, sex, orbody weight as can be readily made by a person skilled in the art.

In one embodiment, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. In one embodiment, one of the ingredients includedin the pharmaceutically acceptable carrier can be for examplepolyethylene glycol (PEG), a biocompatible polymer with a wide range ofsolubility in both organic and aqueous media (Mutter et al. (1979).

In one embodiment, “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In one embodiment, suitable routes of administration, for example,include oral, rectal, transmucosal, transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

In one embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body.

Various embodiments of dosage ranges are contemplated by this invention.The dosage of the polypeptide of the present invention, in oneembodiment, is in the range of 0.05-80 mg/day. In another embodiment,the dosage is in the range of 0.05-50 mg/day. In another embodiment, thedosage is in the range of 0.1-20 mg/day. In another embodiment, thedosage is in the range of 0.1-10 mg/day. In another embodiment, thedosage is in the range of 0.1-5 mg/day. In another embodiment, thedosage is in the range of 0.5-5 mg/day. In another embodiment, thedosage is in the range of 0.5-50 mg/day. In another embodiment, thedosage is in the range of 5-80 mg/day. In another embodiment, the dosageis in the range of 35-65 mg/day. In another embodiment, the dosage is inthe range of 35-65 mg/day. In another embodiment, the dosage is in therange of 20-60 mg/day. In another embodiment, the dosage is in the rangeof 40-60 mg/day. In another embodiment, the dosage is in a range of45-60 mg/day. In another embodiment, the dosage is in the range of 40-60mg/day. In another embodiment, the dosage is in a range of 60-120mg/day. In another embodiment, the dosage is in the range of 120-240mg/day. In another embodiment, the dosage is in the range of 40-60mg/day. In another embodiment, the dosage is in a range of 240-400mg/day. In another embodiment, the dosage is in a range of 45-60 mg/day.In another embodiment, the dosage is in the range of 15-25 mg/day. Inanother embodiment, the dosage is in the range of 5-10 mg/day. Inanother embodiment, the dosage is in the range of 55-65 mg/day.

In one embodiment, the dosage is 20 mg/day. In another embodiment, thedosage is 30 mg/day. In another embodiment, the dosage is 40 mg/day. Inanother embodiment, the dosage is 50 mg/day. In another embodiment, thedosage is 60 mg/day. In another embodiment, the dosage is 70 mg/day. Inanother embodiment, the dosage is 80 mg/day. In another embodiment, thedosage is 90 mg/day. In another embodiment, the dosage is 100 mg/day.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of the desired compound, or compounds, each of which isin one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or inanother embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. Thepharmaceutically-unacceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and crosscarmelose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the pharmaceutical activeingredient as known to one skilled in the art.

Peroral compositions, in some embodiments, comprise liquid solutions,emulsions, suspensions, and the like. In some embodiments,pharmaceutically-acceptable carriers suitable for preparation of suchcompositions are well known in the art. In some embodiments, liquid oralcompositions comprise from about 0.012% to about 0.933% of the desiredcompound or compounds, or in another embodiment, from about 0.033% toabout 0.7%.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments areaqueous solutions or emulsions comprising a safe and effective amount ofthe compounds of the present invention and optionally, other compounds,intended for topical intranasal administration. In some embodiments, hcompositions comprise from about 0.01% to about 10.0% w/v of a subjectcompound, more preferably from about 0.1% to about 2.0, which is usedfor systemic delivery of the compounds by the intranasal route.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intramuscular injection of a liquidpreparation. In some embodiments, liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously, and are thus formulated in a form suitable forintravenous administration. In another embodiment, the pharmaceuticalcompositions are administered intra-arterially, and are thus formulatedin a form suitable for intra-arterial administration. In anotherembodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces, and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the compounds of the present invention arecombined with an additional appropriate therapeutic agent or agents,prepared and applied as solutions, suspensions, or emulsions in aphysiologically acceptable diluent with or without a pharmaceuticalcarrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, pharmaceutical compositions for use in accordancewith the present invention is formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of the active ingredientsinto preparations which, can be used pharmaceutically. In oneembodiment, formulation is dependent upon the route of administrationchosen.

In one embodiment, injectables, of the invention are formulated inaqueous solutions. In one embodiment, injectables, of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In some embodiments, formulations for injection are presentedin unit dosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. In some embodiments, compositions aresuspensions, solutions or emulsions in oily or aqueous vehicles, andcontain formulatory agents such as suspending, stabilizing and/ordispersing agents.

The compositions also comprise, in some embodiments, preservatives, suchas benzalkonium chloride and thimerosal and the like; chelating agents,such as edetate sodium and others; buffers such as phosphate, citrateand acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfite and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In some embodiments, pharmaceutical compositions for parenteraladministration include aqueous solutions of the active preparation inwater-soluble form. Additionally, suspensions of the active ingredients,in some embodiments, are prepared as appropriate oily or water basedinjection suspensions. Suitable lipophilic solvents or vehicles include,in some embodiments, fatty oils such as sesame oil, or synthetic fattyacid esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions contain, in some embodiments, substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In another embodiment, the suspensionalso contain suitable stabilizers or agents which increase thesolubility of the active ingredients to allow for the preparation ofhighly concentrated solutions.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990).

In some embodiments, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use. Compositions are formulated, in someembodiments, for atomization and inhalation administration. In anotherembodiment, compositions are contained in a container with attachedatomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In some embodiments, pharmaceutical compositions suitable for use incontext of the present invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. In some embodiments, a therapeutically effective amount meansan amount of active ingredients effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

The compositions also comprise preservatives, such as benzalkoniumchloride and thimerosal and the like; chelating agents, such as edetatesodium and others; buffers such as phosphate, citrate and acetate;tonicity agents such as sodium chloride, potassium chloride, glycerin,mannitol and others; antioxidants such as ascorbic acid, acetylcystine,sodium metabisulfote and others; aromatic agents; viscosity adjustors,such as polymers, including cellulose and derivatives thereof; andpolyvinyl alcohol and acid and bases to adjust the pH of these aqueouscompositions as needed. The compositions also comprise local anestheticsor other actives. The compositions can be used as sprays, mists, drops,and the like.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. The choice of apharmaceutically-acceptable carrier to be used in conjunction with thecompound is basically determined by the way the compound is to beadministered. If the subject compound is to be injected, in oneembodiment, the pharmaceutically-acceptable carrier is sterile,physiological saline, with a blood-compatible suspending agent, the pHof which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,crosscarmelose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts.) Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

In some embodiments, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Inanother embodiment, the modified compounds exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified compounds. In one embodiment, modificationsalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In another embodiment, the desired in vivo biological activityis achieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In some embodiments, preparation of effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the activeingredients described herein can be determined by standardpharmaceutical procedures in vitro, in cell cultures or experimentalanimals. In one embodiment, the data obtained from these in vitro andcell culture assays and animal studies can be used in formulating arange of dosage for use in human. In one embodiment, the dosages varydepending upon the dosage form employed and the route of administrationutilized. In one embodiment, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. [See e.g., Fingl, et al., (1975) “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical carrier are also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the active ingredient.In one embodiment, the pack, for example, comprise metal or plasticfoil, such as a blister pack. In one embodiment, the pack or dispenserdevice is accompanied by instructions for administration. In oneembodiment, the pack or dispenser is accommodated by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor human or veterinary administration. Such notice, in one embodiment,is labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert.

In one embodiment, it will be appreciated that the polypeptides of thepresent invention can be provided to the individual with additionalactive agents to achieve an improved therapeutic effect as compared totreatment with each agent by itself. In another embodiment, measures(e.g., dosing and selection of the complementary agent) are taken toadverse side effects which are associated with combination therapies.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W.H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Example 1 Generation of EPO constructs Materials and Methods:

Construction of expression vector pCI-dhfr: pCI-neo mammalian expressionvector was purchased from Promega (catalog no. E1841). The vectorcontains a CMV IE enhancer/promoter and neomycin phosphotransferasegene. The pSV2-dhfr clone was purchased from ATCC (Catalog No. 37146).The plasmid contains the murine dhfr gene. The construction of pCI-dhfrvector was performed as follows:

-   -   a. The pSV2-dhfr plasmid was digested with restriction enzyme        BglII (3′ end of the dhfr gene). DNA polymerase I, Large        (Klenow) Fragment was used to fill-in the 5′ overhangs to form        blunt ends. The DNA was then digested with restriction enzyme        AvrII (5′ end of the dhfr gene). The dhfr gene (AvrII-blunt end)        fragment was isolated.    -   b. The pCI-neo vector was digested with restriction enzyme BstXI        (3′ end of the neo gene). DNA polymerase I, Large (Klenow)        Fragment was used to remove the 3′ overhangs to form blunt ends.        The DNA was then digested with restriction enzyme AvrII (5′ end        of the neo gene). The expression vector (AvrII—blunt end) was        isolated.    -   c. The dhfr gene was ligated into pCI vector to form an        expression vector containing the dhfr gene (pCI-dhfr).

Construction of hEPO-CTP variants: A cassette gene containing theC-Terminal peptide (CTP) of the beta subunit of hCG was fused to thecoding sequence of human EPO(NP_(—)000790.2) at different locations.Four EPO-CTP variants were constructed as illustrated in FIGS. 1A-D. TheproEPO signal peptide was used for the construction of the secretedEPO-CTP variants. XbaI-NotI fragments containing Epo sequences wereligated into the pCI-dhfr expression vector of the present invention.

Table 2 hereinbelow summarizes the primer sequences used forconstructing the CTP-containing polypeptides of the present invention.

TABLE 2 SEQ Restriction site Primer ID (underlined in number NO sequencesequence)  1 7 5′ AATCTAGAGGTCATCATGGGGGTGC 3′ XbaI  2 85′ ATTGCGGCCGCGGATCCAGAAGACCTTTATTG 3′ NotI 17^(R) 95′ TAAATATTGGGGTGTCCGAGGGCCC 3′ SspI 10 105′ CCAATATTACCACAAGCCCCACCACGCCTCAT 3′ SspI 11^(R) 115′TGCGGCCGCGGATCCTTATCTGTCCCCTGTCCTGC NotI 3′ 15 12 5′ GCCCTGCTGTCGGAAGC3′  2^(R) 13 5′ ATTGCGGCCGCGGATCCAGAAGACCTTTATTG NotI 23^(R) 145′CTTTGAGGAAGAGGAGCCCAGGACTGGGAGGC3′ 24 15 5′ CCTGGGCTCCTCTTCCTCAAAGGC3′ 28^(R) 16 5′ GCTTCCGACAGCAGGGC 3′

EPO-1 701-1-p17-6 (Epo-1—SEQ ID NO: 1): The XbaI-NotI 702 bp fragmentwas constructed by PCR using the above primers (SEQ ID NOs: 7-16). Thenthe XbaI-NotI PCR fragment containing Epo-ctp sequence was ligated intopCI-dhfr expression vector.

EPO-2 701-2-p24-2 (Epo-2—SEQ ID NO: 2): The XbaI/ApaI fragment (hGH-ctp)of pCI-dhfr-401-2-p21-2 (hGH-ctpx2) was replaced by the XbaI/ApaIfragment (EPO-ctp) of 701-1-p17-6 to create an Epo-ctpx2.

EPO-4-701-4-p42-1(Epo-4—SEQ ID NO: 4): Firstly, a fragment frompCI-dhfr-EPO-ctp (701-1-p17-6) was constructed by PCR using primers 1and 17 followed by XbaI/SspI digestion. This resulted in a fragmentcontaining EPO and partial 5′ CTP.

Secondly, a new fragment was constructed by overlapping PCR, onpGT123-hEpo as a template, using primer 10 and primer 11. SspI/NotIdigestion resulted in fragment containing 3′ partial CTP and Epo.

The two fragments were ligated into pCI-dhfr to construct thep701-4-p42-1 clone.

EPO-3-p56-6 (Epo-3 SEQ ID NO; 3): Primers were purchased fromSigma-Genosys. PCR reaction was performed using primer 15 (SEQ ID NO:12) and primer 2^(R) (SEQ ID NO: 13) and plasmid DNA of pCI-dhfr-EPO-ctpx2 (701-2-p24-2) as a template. As a result of the PCR amplification, a486 bp product was formed and ligated into TA cloning vector(Invitrogen, catalog K2000-01). Stu I-NotI fragment containing *Epo-ctpx2 sequence was isolated (209 bp).

Three sequential PCR reactions were performed. The first reaction wasconducted with primer 1 (SEQ ID NO: 7) and primer 23^(R) (SEQ ID NO: 14)and plasmid DNA of pGT123-epo-ctp as a template; as a result of the PCRamplification, an 80 bp product was formed (signal peptide).

The second reaction was conducted with primer 24 (SEQ ID NO: 15) andprimer 11^(R) (SEQ ID NO: 11) and plasmid DNA of 701-4-p42-1 as atemplate; as a result of the PCR amplification, a 610 bp product wasformed.

The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 11^(R)(SEQ ID NO: 11) and a mixture of the products of the previous tworeactions as a template; as a result of the PCR amplification, a 700 bpproduct was formed and the XbaI-StuI fragment was isolated.

The two fragments (XbaI-StuI and StuI-NotI) were inserted into theeukaryotic expression vector pCI-dhfr (triple ligation) to yield the701-3-p56-6 clone.

EPO-5-p91-4 (Epo-5 SEQ ID NO; 5-(ctp-Epo): Primers were ordered fromSigma-Genosys. A PCR reaction was performed using primer 1 (SEQ ID NO:7) and primer 11^(R) (SEQ ID NO: 11) and plasmid DNA ofpCI-dhfr-ctp-EPO-ctp x2 (701-3-p56-6) as a template; as a result of thePCR amplification, a 670 bp product was formed and ligated into TAcloning vector (Invitrogen, catalog K2000-01). XbaI-NotI fragmentcontaining ctp-Epo sequence was ligated into our eukaryotic expressionvector pCI-dhfr to yield the 701-5-p91-4 clone.

EPO-6-p90-1 (Epo-6 SEQ ID NO: 6-(ctp-Epo-ctp): Three PCR reactions wereperformed. The first reaction was conducted with primer 1 (SEQ ID NO: 7)and primer 38^(R) (SEQ ID NO: 16) and plasmid DNA of 701-3-p56-6 as atemplate; as a result of the PCR amplification, a 400 bp product wasformed.

The second reaction was conducted with primer 15 (SEQ ID NO: 12) andprimer 2^(R) (SEQ ID NO: 13) and plasmid DNA of 701-1-p17-6 as atemplate; as a result of the PCR amplification, a 390 bp product wasformed.

The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 2^(R)(SEQ ID NO: 13) and a mixture of the products of the previous tworeactions as a template; as a result of the PCR amplification, a 787 bpproduct was formed and ligated into TA cloning vector (Invitrogen,catalog K2000-01). The XbaI-NotI fragment containing ctp-Epo-ctpsequence was ligated into the eukaryotic expression vector pCI-dhfr toyield the 701-6-p90-1 clone.

Example 2

Expression and Isolation of EPO-CTP Polypeptides

Materials and Methods

DNA transfection and clone selection: DG44 cells were transfected withpCI-DHFR expression vectors containing EPO-CTP variants using FuGENE6Reagent (FuGENE Transfection Reagent—Roche Cat. 11 815 091 001). 48 hrfollowing transfection, cells were diluted and seeded at 50-200 cellsper well in a selective medium (CD DG44 Medium w/o HT (Gibco: Scotlandpart: #07990111A) Sku num.: ME060027 supplemented with 8 mM L-GlutamineBiological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68solution (Gibco: Cat: 240040-032). Selected clones were screened forhighest protein production using commercial ELISA. 3-5 producing clonesper each variant were frozen for a backup cell bank. A selected clonefor each variant was adapted to growth in larger scale cultures up to ILflasks on an orbital shaker platform. Supernatants were collected andanalyzed by ELISA, SDS-PAGE and western blot. Following the withdrawalof aliquots, the protein-containing supernatants were kept frozen untilfurther use.

Cell culture: DG44 cells were maintained in DG44 medium with HT (cat#12610-010, Gibco) supplemented with 8 mM L-Glutamine (BiologicalIndustries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution(Gibco: Cat: 240040-032), at 37° C. in humidified 8% CO₂ incubator.Transfected clones were maintained in DG44 basal medium without HTsupplement, hypoxanthine and thymidine, with pluronic acid andL-glutamine.

Sample preparation: Supernatants were collected, filtrated and analyzedby ELISA to determine protein concentration. SDS-PAGE and western blotwere used to determine purity and identity. Following ELISA, sampleconcentrations were defined and the solution was dialyzed against PBS.Following the withdrawal of aliquots, the protein-contained supernatantswere kept frozen at −20° C. until further use.

Western Blotting Samples were electrophoresed on nondenaturing 15%SDS-polyacrylamide gels. Gels were allowed to equilibrate for 10 min in25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol). Proteins weretransferred to a 0.2 μm pore size nitrocellulose membrane (Sigma, SaintLouis, Mo.) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresiscell (Biorad Laboratories, Richmond, Calif.). The nitrocellulosemembrane was incubated in 5% non-fat dry milk for 2 h at roomtemperature. The membrane was incubated with EPO anti-serum (1:1000titer) overnight at 4° C. followed by three consecutive washes in PBScontaining 0.1% Tween (10 min/wash). The membrane was incubated withsecondary antibody conjugated to Horse Radish Peroxidase (HRP) (Zymed,San Francisco, Calif.) for 2 h at room temperature, followed by threewashes. Finally, the nitrocellulose paper was reacted with enhancedchemiluminescent substrate (ECL) (Pierce, Rockford, Ill.) for 5 min,dried with a Whatman sheet, and exposed to X-ray film.

Results

Table 3 hereinbelow shows the concentrations of the various CTP-modifiedEPO forms obtained from 5 selected clones and their preparation forfurther testing.

TABLE 3 Stock Post dilution in Post Titer Mock sup. accordingultrafiltration #Version # Clone IU/ml¹ to Epo3 titer IU/ml² IU/ml³ Epo017 3093 102 335 SEQ ID NO: 16 Epo1 47 1049 104 291 SEQ ID NO: 1 Epo2 672160 110 303 SEQ ID NO: 2 Epo3 85 105 119 392 SEQ ID NO: 3 Epo4 112 6100ND 342 SEQ ID NO: 4 ¹EPO variants stock concentration were determined byELISA (Quantikine IVD Epo ELISA, DEP00, R&D Systems) ²Samples EPO-0, 1,2 and 4 were diluted to 105 IU/ml in mock sup (Adjusted to Epo3 titer).Epo0 = wild type EPO expressed in the same system as the CTP modifiedEPOs ³All samples were concentrated and dialyzed by ultrafiltrationagainst PBS to a final concentration of 180 IU/ml.

All proteins were detected by Western blot as illustrated in FIG. 2.

Example 3 Biological Activity of the EPO-CTP Polypeptides of the PresentInvention

The TF-1 bioactivity test represents the ability of the EPO-CTP variantsto bind its receptor and then stimulate activity which results in cellproliferation. Therefore, this test was used as a first step inevaluating the biological potency of the EPO-CTP polypeptides of thepresent invention.

Materials and Methods

Cell Proliferation Analysis: Proliferation assay was performed with thecell line TF-1, measuring levels of MTT cellular stain as a function ofEPO activity (Kitamura et al., Kitamura, T. et al. (1989) Establishmentand characterization of a unique human cell line that proliferates;Hammerling U. et al. In vitro bioassay for human erythropoietin based onproliferative stimulation of an erythroid cell line and analysis ofcarbohydrate-dependent microheterogeneity. Journal of Pharm. Biomed.Analysis 14(11): 1455-1469 (1996). Exponentially growing TF-1 cells werewashed twice, plated at about 10⁴ cells/well in microtiter plates, andincubated in basal medium with a titrated dilution series of EPO(Recormon), EPO standard (NIBSC standard), rhEPO (MOD-7010), MOD-701variants (EPO-1, EPO-2, EPO-3 and EPO-4) for 48 hours. 4 hours prior toassaying for cell proliferation, MTT reagent was added to the wells, andabsorbance was measured by ELISA reader. A calculated proteinconcentration value for each variant protein was obtained from Eprex's(Epoetin (EPO)-man-made form of the human hormone) dose-responsestandard curve.

Results

The in vitro biological activity of EPO polypeptides was determined withan Epo-dependent cell line, human erythroleukemia TF-1 (DSMZ Cell Bank)[Dong et al., Biochemical and Biophysical Research Communications,Volume 339, Issue 1, 6 Jan. 2006, Pages 380-385]. The MTT assay wasperformed [Hammerling U. et al. In vitro bioassay for humanerythropoietin based on proliferative stimulation of an erythroid cellline and analysis of carbohydrate-dependent microheterogeneity. Journalof Pharm. Biomed. Analysis 14(11): 1455-1469 (1996);], and thelaboratory standard of EPO used to generate the standard curve wascalibrated against the International Standard (Epo ampoule code 87/684of NIBSC).

The results are summarized in Table 4 hereinbelow. The results indicatethat the highest potency was achieved by using EPO 3 and EPO 0 in both 2and 0.5 IU/ml concentrations.

TABLE 4 Eprex TF-1 Bioactivity IU/ml STD EPO 0 EPO 1 EPO 2 EPO 3 EPO 4IU/ SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID EPO ml NO: 16 NO: 1 NO: 2 NO: 3NO: 4 Recormon st 2 4.93 2.32 2.13 6.91 3.55 3.44 7.40 0.5 1.60 0.760.53 1.34 0.84 0.87 1.53

Conclusion

As summarized in Table 4 hereinabove, different levels of potency wereexerted by EPO-CTP polypeptides, indicating differences in receptorbinding. EPO-CTP polypeptides differ by the number of CTP cassettes andthe location to which they are fused. EPO-1 and EPO-2 contain 1 CTPsequence or 2 CTP sequences at the C-terminal of EPO, while EPO-3contains 1 CTP at N-terminal and 2 CTP sequences at C-terminal. EPO-4 isa dimer of two EPO molecules linked by CTP sequence. EPO-3 demonstratedpotency level quite similar to WT-EPO, while EPO-1 and EPO-4 were about50% less potent than WT-EPO, and EPO-2 potency was even less than 50%.

Example 4 Evaluation of the EPO-CTP Polypeptides of the PresentInvention in a Mouse Model

The following experiment was performed in order to compare thebio-activity of the EPO-CTP polypeptides of the present invention andcommercial EPO

Materials and Methods

Animals: Species/Strain: ICR or CD-1 Mice of either sex about 20-25 gGroup Size: n = 7 No. Groups: 9 Total No. Animals: n = 63

Experimental design of the study: The experiment was set up assummarized in Table 5 hereinbelow.

TABLE 5 No. Mice TREATMENT Dosing Group No. per Group Compound DoseLevel Regimen 1 n = 7 Vehicle (Control) 0 1× weekly 2 MOCK 3 MOD-7010 15μg/kg 4 MOD-7011 5 MOD-7012 6 MOD-7013 7 MOD-7014 8 Commercial 15 μg/kg9 rhEPO  5 μg/kg 3× weekly

Animal treatment: All animals were administered with either control orthe test EPO polypeptides of the present invention by bolus injection.The injection volume did not exceed 10 ml/kg. The length of theexperiment was 22 days. A morbidity and mortality check was performeddaily.

Reticulocyte count and hematocrit (hct) examination: Reticulocyte countwas carried out in all test animals at day 2 and 14 hrs following the1st respective vehicle or treatment injection. HCT was determined in allanimals once prior to initial treatment (“0” Baseline control) and at 24hrs after the 1st respective vehicle or treatment injection, andthereafter twice weekly until study termination (Day-22).

Results

The hematocrit results which are illustrated in FIGS. 3-5 show that EPO3 has the highest hematocrit percentage change from baseline compared toEPO 1, EPO 2, Recormon 1, Recormon 3, rhEPO, and vehicle. The resultsdemonstrating the percentage of reticulocytes in mice treated with theEPO-CTP polypeptides are summarized in Table 6 hereinbelow. Theseresults show that EPO-3 is the most potent stimulator of erythropoiesis.

TABLE 6 % reticulocytes Days 2 14 Control 3.72 3.46 1.08 0.8 Mock 3.53.64 0.6 1.13 7010 SEQ ID NO: 16 3.5 3.9 0.6 1.54 7011 SEQ ID NO: 1 3.521.94 1.38 1.08 7012 SEQ ID NO: 2 3.82 3.0 1.02 0.88 7013 SEQ ID NO: 32.66 5.20 0.97 2.96 7014 SEQ ID NO: 4 3.48 3.82 0.71 0.90 Recormon 1/W3.23 3.27 0.73 0.59 Recormon 3/w 4.13 4.24 1.21 1.14

Conclusion

The in vivo experiment was designed to measure two parameters; the firstwas to measure erythropoiesis parameters such as percentage ofreticulocytes and increase of hemoglobin, RBC and hematocrit levels. Thesecond was to measure the durability of the biological activity of eachvariant by injecting once weekly doses.

A superior performance of EPO-3 in its ability to stimulateerythropoiesis was observed in normal mice.

Example 5 Comparison of the EPO-CTP Polypeptides of the PresentInvention to Aranesp

The following experiment was performed in order to compare thebiological activity of a single bolus dose of some EPO-CTP polypeptidesof the present invention, commercial EPO and Aranesp. Aranesp is acommercial long-acting recombinant erythropoietin in which two sitemutations were introduced, resulting in two additional N-glycosylationsites and an increase in the number of incorporated sialic acidresidues.

Materials and Methods Animals:

Species/Strain: Female CD-1 Mice of either sex about 20-25 gGroup Size: n=3

Experimental design of the study: The experiment was set up assummarized in Table 7 hereinbelow.

TABLE 7 Dose animals/ Solution Dose group/ Conc. Volume Time-Points*Group # Test Article time-point (μg/mL) (mL/kg) (hourspost-administration) 1 MOD-7010 3 1.5 10 0 (Pre-dose), 0.25, 0.5, 1, 2,6, SEQ ID NO: 11 24, 48, 96, 168, 216, 264 and 336 hr post-doseadministration 2 MOD-7013 3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, SEQID NO: 3 168, 216, 264 and 336 hr post- dose administration 3 Aranesp 31.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, 168, 216, 264 and 336 hr post-dose administration

Animal treatment: All animals were administered with either control orthe test EPO polypeptides of the present invention by bolus injection.The injection volume did not exceed 10 ml/kg. The length of theexperiment was 14 days. A morbidity and mortality check was performeddaily.

Reticulocyte count and hematocrit (hct) examination: Reticulocyte countand hematocrit examination were performed as described above.

Results

The results are illustrated in FIGS. 6-9. Following a single I.V.injection of 15 μg/kg of EPO 3, all three blood parameters associatedwith erythropoietin i.e. number of reticulocytes, hemoglobin level andhematocrit, were improved relative to those obtained with similarinjected dose of rhEPO or Aranesp.

Example 6 Comparison of the Pharmacokinetics of EPO-CTP Polypeptides ofthe Present Invention to Aranesp

The following experiment was performed in order to compare thepharmacokinetics of EPO-CTP polypeptide of the present invention,commercial EPO and Aranesp.

Materials and Methods

Serum samples were analyzed in order to determine specific concentrationlevels for each sample. Concentration and time-point data were processedusing WinNonLin noncompartmental analysis. Parameters determinedincluded: AUC, CL, Ke, t1/2, Cmax, T_(max), and Vdz.

Serum concentrations were determined using two ELISA kits in parallel.EPO-3 serum concentration was measured using StemCell ELISA kit incomparison to EPO-0 and Aranesp serum concentration which weredetermined using R&D system ELISA kit.

Results

The results of the pharmacokinetic analysis are summarized in Table 8,hereinbelow. These results show that EPO 3 exhibited favorablepharmacokinetic measures as indicated for example in AUC measures, t1/2,and Cmax. Tmax measures were equal to EPO-0, EPO-3, and Aranesp.

TABLE 8 Parameters Units EPO-0 EPO-3 Aranesp AUClast hr*mIU/mL 31739306072 178661 CL{circumflex over ( )} mL/hr/kg 1.1152 0.2188 0.1207 Ke1/hr 0.157 0.0529 0.0639 t½ hr 4.4139 13.1141 10.84 Cmax mIU/mL 1076616466 13266 Tmax Hr 0.25 0.25 0.25 Vdz mL/kg 7.1017 4.1394 1.8877

The results of the serum concentration analysis are illustrated in FIG.9. These results show that EPO-3 was still detectable in the serum afterabout 190 hours. Both EPO-0 and Aranesp were not detectable in the serumafter about 140 hours and 50 hours, respectively.

Conclusion

Clearance of EPO-3 (MOD-7013) from the blood of CD-1 mice wassignificantly slower than that for rhEPO or Aranesp. The correspondingcalculated half life times were: rhEPO—4.41 h; Aranesp—0.84 h; andMOD-7013-13.11 h.

Example 7 Generation of hGH Constructs Materials and Methods

Four hGH clones (variants of 20 kD protein) were synthesized. Xba I-NotI fragments containing hGH sequences from the four variants were ligatedinto the eukaryotic expression vector pCI-dhfr previously digested withXbaI-NotI. DNA from the 4 clones (401-0, 1, 2, 3 and 4) was prepared.Another partial hGH clone (1-242 bp) from 22 kD protein was alsosynthesized (0606114). Primers were ordered from Sigma-Genosys. Theprimer sequences used to generate the hGH-CTP polypeptides of thepresent invention are summarized in Table 9, hereinbelow.

TABLE 9 SEQ Restriction site Primer ID (underlined in number NO sequencesequence) 25 27 5′ CTCTAGAGGACATGGCCAC XbaI 3′ 32^(R) 285′ ACAGGGAGGTCTGGGGGTTCT GCA 3′ 33 29 5′ TGCAGAACCCCCAGACCTCCC TGTGC 3′ 4^(R) 30 5′ CCAAACTCATCAATGTATCTT A 3′ 25 31 5′ CTCTAGAGGACATGGCCACXbaI 3′ 35^(R) 32 5′ CGAACTCCTGGTAGGTGTCAA AGGC 3′ 34 335′ GCCTTTGACACCTACCAGGAG TTCG 3′ 37^(R) 34 5′ACGCGGCCGCATCCAGACCTTC NotIATCACTGAGGC3′ 39^(R) 35 5′ GCGGCCGCGGACTCATCAGAAGCC GCAGCTGCCC3′

Construction of 402-0-p69-1 (hGH) SEQ ID NO: 36: MOD-4020 is the wildtype recombinant human growth hormone (without CTP) which was preparedfor use as control in the below described experiments.

Three PCR reactions were performed. The first reaction was conductedwith primer 25 and primer 32^(R) and plasmid DNA of 0606114 (partialclone of hGH 1-242 bp) as a template; as a result of the PCRamplification, a 245 bp product was formed.

The second reaction was conducted with primer 33 and primer 4^(R) andplasmid DNA of 401-0-p57-2 as a template; as a result of the PCRamplification, a 542 bp product was formed.

The last reaction was conducted with primers 25 and 4^(R) and a mixtureof the products of the previous two reactions as a template; as a resultof the PCR amplification, a 705 bp product was formed and ligated intothe TA cloning vector (Invitrogen, catalog K2000-01). The XbaI-NotIfragment containing hGH-0 sequence was ligated into the eukaryoticexpression vector pCI-dhfr. The vector was transfected into DG-44 CHOcells. Cells were grown in protein-free medium.

Construction of 402-1-p83-5 (hGH-CTP)—SEQ ID NO: 37 and402-2-p72-3(hGH-CTPx2)-SEQ ID NO: 38: MOD-4021 is a recombinant humangrowth hormone which was fused to 1 copy of the C-terminal peptide ofthe beta chain of human Chorionic Gonadotropin (CTP). The CTP cassetteof MOD-4021 was attached to the C-terminus (one cassette). MOD-4022 is arecombinant human growth hormone which was fused to 2 copies of theC-terminal peptide of the beta chain of human Chorionic Gonadotropin(CTP). The two CTP cassettes of MOD-4022 were attached to the C-terminus(two cassettes).

Construction of hGH-CTP and hGH-CTP-CTP was performed in the same way asthe construction of hGH-0. pCI-dhfr-401-1-p20-1 (hGH*-ctp) andpCI-dhfr-401-2-p21-2 (hGH*-ctp x2) were used as templates in the secondPCR reaction.

MOD-4021 and MOD-4022 were expressed in DG-44 CHO cells. Cells weregrown in protein-free medium. The molecular weight of MOD-4021 is ˜30.5Kd since hGH has a MW of 22 Kd while each “CTP cassette” contributes 8.5Kd to the overall molecular weight (see FIG. 10). The molecular weightof MOD-4022 is 39 Kd (see FIG. 10).

Construction of 402-3-p81-4 (CTP-hGH-CTP-CTP)—SEQ ID NO: 39 and402-4-p82-9(CTP*hGH-CTP-CTP)—SEQ ID NO: 40: MOD-4023 is a recombinanthuman growth hormone which was fused to 3 copies of the C-terminalpeptide of the beta chain of human Chorionic Gonadotropin (CTP). Thethree CTP cassettes of MOD-4023 were attached to both N-terminus (onecassette) and the C-terminus (two cassettes). MOD-4024 is a recombinanthuman growth hormone which is fused to 1 truncated and 2 complete copiesof the C-terminal peptide of the beta chain of human ChorionicGonadotropin (CTP). The truncated CTP cassette of MOD-4024 was attachedto the N-terminus and two CTP cassettes were attached to the C-terminus(two cassettes).

Three PCR reactions were performed. The first reaction was conductedwith primer 25 and primer 35^(R) and plasmid DNA of p401-3-p12-5 or401-4-p22-1 as a template; as a result of the PCR amplification, a 265or 220 bp product was formed. The second reaction was conducted withprimer 34 and primer 37^(R) and plasmid DNA of TA-hGH-2-q65-1 as atemplate; as a result of the PCR amplification, a 695 bp product wasformed. The last reaction was conducted with primers 25 and 37^(R) and amixture of the products of the previous two reactions as a template; asa result of the PCR amplification, a 938 or 891 bp product was formedand ligated into TA cloning vector (Invitrogen, catalog K2000-01). XbaI-Not I fragment containing hGH sequence was ligated into our eukaryoticexpression vector pCI-dhfr.

MOD-4023 and MOD-4024 were expressed in DG-44 CHO cells. Cells weregrown in protein-free medium. The molecular weight of MOD-4023 is ˜47.5Kd (see FIG. 10) and the molecular weight of MOD-4024 is ˜43.25 Kd (seeFIG. 10).

Construction of 402-6-p95a-8 (CTP-hGH-CTP)—SEQ ID NO: 41: Constructionof hGH-6 was performed in the same way as the construction of hGH-3.pCI-dhfr-402-1-p83-5 (hGH-ctp) was used as a template in the second PCRreaction.

Construction of 402-5-p96-4 (CTP-hGH)—SEQ ID NO: 42: PCR reaction wasperformed using primer 25 and primer 39^(R) and plasmid DNA ofpCI-dhfr-ctp-EPO-ctp (402-6-p95a-8) as a template; as a result of thePCR amplification, a 763 bp product was formed and ligated into TAcloning vector (Invitrogen, catalog K2000-01). Xba 1-Not I fragmentcontaining ctp-hGH sequence was ligated into our eukaryotic expressionvector pCI-dhfr to yield 402-5-p96-4 clone.

Example 8

In Vivo Bioactivity Tests of hGH-CTP Polypeptides of the PresentInvention

The following experiment was performed in order to test the potentiallong acting biological activity of hGH-CTP polypeptides in comparisonwith commercial recombinant human GH and MOD-4020.

Materials and Methods

Female hypophysectomized rats (60-100 g) received a weekly S.C.injection of 21.7 μg hGH-CTP polypeptides or a once daily 5 μg S.C.injection of control commercial rhGH.

Weight was measured in all animals before treatment, 24 hours followingfirst injection and then every other day until the end of the study onday 21. Each point represents the group's average weight gain percentage((Weight day 0−weight last day)/Weight day 0). Average weight gain wasnormalized against once-daily injection of commercial hGH. The treatmentschedule is summarized in Table 10.

TABLE 10 Equimolar Accumulate Treatment Dose Dosage Dose No. Drug NRoute Schedule (μg/rat) (μg/rat) Vol. (ml) 1 Vehicle 7 s.c. days 1, 7 NANA 0.25 and 13; 1/W 2 Mock 7 s.c days 1, 7 NA NA 0.25 and 13; 1/W 3MOD-4020 7 s.c days 1, 7 21.7 65 0.25 SEQ ID NO: 36 and 13; 1/W 4MOD-4021 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 37 and 13; 1/W 5MOD-4022 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 38 and 13; 1/W 6MOD-4023 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 39 and 13; 1/W 7MOD-4024 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 40 and 13; 1/W 8Commercial 7 s.c. days 1, 7 21.7 65 0.25 hGH v.1 and 13; 1/W 9Commercial 7 s.c. days 1-13; 5 65 0.25 hGH v.1 d/W

Results

Results are summarized in FIG. 11. These results show that MOD-4023 (SEQID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced over 120% weight gaincompared to commercial rhGH which induced 100% weight gain.

CONCLUSION

3 weekly doses (Days of injections; 1, 7, and 13) of 21.7 μg of MOD-4023(SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced a 30% greaterweight increase in hypophysectomised rats compared to commercial rhGHinjected at the same accumulated dose which was administered once perday at a dose of 5 μg for 13 days.

1. A polypeptide consisting of a non-human EPO polypeptide, onechorionic gonadotrophin carboxy terminal peptide attached to the aminoterminus of said non-human EPO polypeptide, and two chorionicgonadotrophin carboxy terminal peptides attached to the carboxy terminusof said non-human EPO polypeptide.
 2. The polypeptide of claim 1,wherein the sequence of at least a single chorionic gonadotrophincarboxy terminal peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:17 and SEQ ID NO:18.
 3. Thepolypeptide of claim 1, wherein at least a single chorionicgonadotrophin carboxy terminal peptide is truncated. 4-8. (canceled) 9.A polypeptide consisting a non-human EPO polypeptide, one chorionicgonadotrophin carboxy terminal peptide attached to the amino terminus ofsaid non-human EPO polypeptide, two chorionic gonadotrophin carboxyterminal peptides attached to the carboxy terminus of said non-human EPOpolypeptide, and a signal peptide attached to the amino terminus of saidone chorionic gonadotrophin carboxy terminal peptide.
 10. Thepolypeptide of claim 9, wherein the sequence of said signal peptide isas set forth in SEQ ID NO:
 19. 11. A composition comprising thepolypeptide of claim 1 and a pharmaceutically acceptable carrier.
 12. Amethod of improving a biological half life of a non-human EPOpolypeptide, comprising the step of attaching one chorionicgonadotrophin carboxy terminal peptide to the amino terminus of saidnon-human EPO polypeptide and two chorionic gonadotrophin carboxyterminal peptides to the carboxy terminus of said non-human EPOpolypeptide, thereby improving a biological half life of a non-human EPOpolypeptide.
 13. A method of improving a biological activity of anon-human EPO polypeptide, comprising the step of attaching a firstchorionic gonadotrophin carboxy terminal peptide to an amino terminus ofsaid non-human EPO polypeptide and a second chorionic gonadotrophincarboxy terminal peptide to a carboxy terminus of said non-human EPOpolypeptide, thereby improving a biological activity of a non-human EPOpolypeptide.
 14. The method of claim 13, further comprising the step ofattaching a third chorionic gonadotrophin carboxy terminal peptide intandem to said second chorionic gonadotrophin carboxy terminal peptide.15. The method of claim 13, wherein the sequence of at least a singlechorionic gonadotrophin carboxy terminal peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:17 and SEQ IDNO:18.
 16. The method of claim 13, wherein at least a single chorionicgonadotrophin carboxy terminal peptide is truncated.
 17. The method ofclaim 13 wherein said improving a biological activity further comprisesimproving biological half life of said non-human EPO polypeptide. 18-24.(canceled)
 25. A method of treating or reducing an incidence of anemiain a subject, comprising the step of administering to said subject atherapeutically effective amount of a non-human EPO polypeptide havingan improved biological activity, wherein said non-human EPO polypeptidehaving an improved biological activity comprises a non-human EPOpolypeptide and at least two chorionic gonadotrophin carboxy terminalpeptides, wherein a first chorionic gonadotrophin carboxy terminalpeptide of said at least two chorionic gonadotrophin carboxy terminalpeptides is attached to an amino terminus of said EPO polypeptide, and asecond chorionic gonadotrophin carboxy terminal peptide of said at leasttwo chorionic gonadotrophin carboxy terminal peptides is attached to acarboxy terminus of said EPO polypeptide, thereby treating or reducingan incidence of anemia in a subject.
 26. The method of claim 25, whereinsaid EPO polypeptide having an improved biological activity furthercomprises a third chorionic gonadotrophin carboxy terminal peptideattached in tandem to said second chorionic gonadotrophin carboxyterminal peptide
 27. The method of claim 25, wherein the sequence of atleast a single chorionic gonadotrophin carboxy terminal peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 17 and SEQ ID NO:18.
 28. The method of claim 25, wherein atleast a single chorionic gonadotrophin carboxy terminal peptide istruncated.
 29. A polynucleotide molecule comprising a coding portion,wherein said coding portion consists the DNA sequence encoding thepolypeptide of claim
 1. 30. A polynucleotide molecule comprising acoding portion, wherein said coding portion consists the DNA sequenceencoding the polypeptide of claim
 9. 31. The polynucleotide of claim 30,wherein the sequence of said signal peptide is as set forth in SEQ IDNO:
 19. 32. An expression vector comprising the polynucleotide of claim29.
 33. An expression vector comprising the polynucleotide of claim 30.34. A cell comprising the expression vector of claim
 32. 35. A cellcomprising the expression vector of claim
 33. 36. A compositioncomprising the expression vector of claim
 32. 37. A compositioncomprising the expression vector of claim
 33. 38-43. (canceled)
 44. Amethod of producing a non-human EPO polypeptide with an improvedbiological half life and an improved biological activity in a cell,comprising the step of transfecting said cell with an expression vectorof claim 32 or claim
 33. 45-47. (canceled)